WorldWideScience

Sample records for bzip transcriptional activator

  1. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor

    KAUST Repository

    Nagashima, Yukihiro

    2011-07-01

    IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.

  2. Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

    Science.gov (United States)

    Ciaccio, Natalie A; Reynolds, T Steele; Middaugh, C Russell; Laurence, Jennifer S

    2012-11-01

    Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation. PMID:23067245

  3. Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

    Science.gov (United States)

    Ciaccio, Natalie A; Reynolds, T Steele; Middaugh, C Russell; Laurence, Jennifer S

    2012-11-01

    Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation.

  4. Two domains of the human bZIP transcription factor TCF11 are necessary for transactivation

    OpenAIRE

    Murphy, Paula

    2001-01-01

    PUBLISHED TCF11 is a bZIP transcription factor of the CNC subfamily. It has been implicated in the regulation of the antioxidant response and is vital during embryonic development, but its precise biological functions have not yet been fully worked out. Structural characterization of the gene and several of its products indicates that complex regulatory mechanisms are employed. To investigate how altering the structure of the gene products might influence their activity we have mapped func...

  5. Expression analysis of bZIP transcription factor encoding genes in response to water deficit stress in rice.

    Science.gov (United States)

    Ali, Kishwar; Rai, R D; Tyagi, Aruna

    2016-05-01

    In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate several developmental processes and activate genes in response to biotic and abiotic stresses. Role of stress responsive bZIP transcription factors was studied in paddy in relation to different stages of development and water deficit stress (WDS) in a drought tolerant cultivar N22 and susceptible IR 64. Further, relative water content (RWC), membrane stability index (MSI) and abscisic acid (ABA) content were measured as indices of WDS at different stages of development and levels of stress. Expression of stress responsive bZIP transcription factors was directly correlated to developmental stage and WDS and indirectly to RWC, MSI and ABA content. PMID:27319052

  6. Genome-wide characterization and analysis of bZIP transcription factor gene family related to abiotic stress in cassava.

    Science.gov (United States)

    Hu, Wei; Yang, Hubiao; Yan, Yan; Wei, Yunxie; Tie, Weiwei; Ding, Zehong; Zuo, Jiao; Peng, Ming; Li, Kaimian

    2016-01-01

    The basic leucine zipper (bZIP) transcription factor family plays crucial roles in various aspects of biological processes. Currently, no information is available regarding the bZIP family in the important tropical crop cassava. Herein, 77 bZIP genes were identified from cassava. Evolutionary analysis indicated that MebZIPs could be divided into 10 subfamilies, which was further supported by conserved motif and gene structure analyses. Global expression analysis suggested that MebZIPs showed similar or distinct expression patterns in different tissues between cultivated variety and wild subspecies. Transcriptome analysis of three cassava genotypes revealed that many MebZIP genes were activated by drought in the root of W14 subspecies, indicating the involvement of these genes in the strong resistance of cassava to drought. Expression analysis of selected MebZIP genes in response to osmotic, salt, cold, ABA, and H2O2 suggested that they might participate in distinct signaling pathways. Our systematic analysis of MebZIPs reveals constitutive, tissue-specific and abiotic stress-responsive candidate MebZIP genes for further functional characterization in planta, yields new insights into transcriptional regulation of MebZIP genes, and lays a foundation for understanding of bZIP-mediated abiotic stress response. PMID:26947924

  7. The phylogeny of C/S1 bZIP transcription factors reveals a shared algal ancestry and the pre-angiosperm translational regulation of S1 transcripts.

    Science.gov (United States)

    Peviani, Alessia; Lastdrager, Jeroen; Hanson, Johannes; Snel, Berend

    2016-01-01

    Basic leucine zippers (bZIPs) form a large plant transcription factor family. C and S1 bZIP groups can heterodimerize, fulfilling crucial roles in seed development and stress response. S1 sequences also harbor a unique regulatory mechanism, termed Sucrose-Induced Repression of Translation (SIRT). The conservation of both C/S1 bZIP interactions and SIRT remains poorly characterized in non-model species, leaving their evolutionary origin uncertain and limiting crop research. In this work, we explored recently published plant sequencing data to establish a detailed phylogeny of C and S1 bZIPs, investigating their intertwined role in plant evolution, and the origin of SIRT. Our analyses clarified C and S1 bZIP orthology relationships in angiosperms, and identified S1 sequences in gymnosperms. We experimentally showed that the gymnosperm orthologs are regulated by SIRT, tracing back the origin of this unique regulatory mechanism to the ancestor of seed plants. Additionally, we discovered an earlier S ortholog in the charophyte algae Klebsormidium flaccidum, together with a C ortholog. This suggests that C and S groups originated by duplication from a single algal proto-C/S ancestor. Based on our observations, we propose a model wherein the C/S1 bZIP dimer network evolved in seed plants from pre-existing C/S bZIP interactions. PMID:27457880

  8. Coupling of folding and DNA-binding in the bZIP domains of Jun-Fos heterodimeric transcription factor.

    Science.gov (United States)

    Seldeen, Kenneth L; McDonald, Caleb B; Deegan, Brian J; Farooq, Amjad

    2008-05-01

    In response to mitogenic stimuli, the heterodimeric transcription factor Jun-Fos binds to the promoters of a diverse array of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryogenic development and cancer. In so doing, Jun-Fos heterodimer regulates gene expression central to physiology and pathology of the cell in a specific and timely manner. Here, using the technique of isothermal titration calorimetry (ITC), we report detailed thermodynamics of the bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of the bZIP domains to both TRE and CRE is under enthalpic control and accompanied by entropic penalty at physiological temperatures. Although the bZIP domains bind to both TRE and CRE with very similar affinities, the enthalpic contributions to the free energy of binding to CRE are more favorable than TRE, while the entropic penalty to the free energy of binding to TRE is smaller than CRE. Despite such differences in their thermodynamic signatures, enthalpy and entropy of binding of the bZIP domains to both TRE and CRE are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the free energy of binding. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change determined is much larger than that expected from the direct association of bZIP domains with DNA. This observation is interpreted to suggest that the basic regions in the bZIP domains are largely unstructured in the absence of DNA and only become structured upon interaction with DNA in a coupled folding and binding manner. Our new findings are rationalized in the context of 3D structural models of bZIP domains of Jun-Fos heterodimer in complex with dsDNA oligos containing the TRE and CRE consensus sequences. Taken together, our study demonstrates that enthalpy is

  9. Genome-Wide Identification and Characterization of bZIP Transcription Factors in Brassica oleracea under Cold Stress.

    Science.gov (United States)

    Hwang, Indeok; Manoharan, Ranjith Kumar; Kang, Jong-Goo; Chung, Mi-Young; Kim, Young-Wook; Nou, Ill-Sup

    2016-01-01

    Cabbages (Brassica oleracea L.) are an important vegetable crop around world, and cold temperature is among the most significant abiotic stresses causing agricultural losses, especially in cabbage crops. Plant bZIP transcription factors play diverse roles in biotic/abiotic stress responses. In this study, 119 putative BolbZIP transcription factors were identified using amino acid sequences from several bZIP domain consensus sequences. The BolbZIP members were classified into 63 categories based on amino acid sequence similarity and were also compared with BrbZIP and AtbZIP transcription factors. Based on this BolbZIP identification and classification, cold stress-responsive BolbZIP genes were screened in inbred lines, BN106 and BN107, using RNA sequencing data and qRT-PCR. The expression level of the 3 genes, Bol008071, Bol033132, and Bol042729, was significantly increased in BN107 under cold conditions and was unchanged in BN106. The upregulation of these genes in BN107, a cold-susceptible inbred line, suggests that they might be significant components in the cold response. Among three identified genes, Bol033132 has 97% sequence similarity to Bra020735, which was identified in a screen for cold-related genes in B. rapa and a protein containing N-rich regions in LCRs. The results obtained in this study provide valuable information for understanding the potential function of BolbZIP transcription factors in cold stress responses. PMID:27314020

  10. Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage.

    Directory of Open Access Journals (Sweden)

    Yili Bai

    Full Text Available bZIP (basic leucine zipper transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5. Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14 and BrABI5b (Bra017251/BrbZIP13, are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy or LUC (LUCIFERASE expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.

  11. Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage

    Science.gov (United States)

    Hu, Xiaochen; Sun, Congcong; Li, Yanlin; Wang, Dandan; Wang, Qinhu; Pei, Guoliang; Zhang, Yanfeng; Guo, Aiguang; Zhao, Huixian; Lu, Haibin; Mu, Xiaoqian; Hu, Jingjiang; Zhou, Xiaona; Xie, Chang Gen

    2016-01-01

    bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage. PMID:27414644

  12. Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage.

    Science.gov (United States)

    Bai, Yili; Zhu, Wenbo; Hu, Xiaochen; Sun, Congcong; Li, Yanlin; Wang, Dandan; Wang, Qinhu; Pei, Guoliang; Zhang, Yanfeng; Guo, Aiguang; Zhao, Huixian; Lu, Haibin; Mu, Xiaoqian; Hu, Jingjiang; Zhou, Xiaona; Xie, Chang Gen

    2016-01-01

    bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (Brassica rapa) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in Brassica rapa (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like BrABI5a (Bra005287/BrbZIP14) and BrABI5b (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven HIS (a HIS3 reporter gene, which confers His prototrophy) or LUC (LUCIFERASE) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis abi5-1 is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage. PMID:27414644

  13. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    Science.gov (United States)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  14. Stress sensing in plants by the ER stress sensor/transducer, bZIP28

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    Renu eSrivastava

    2014-02-01

    Full Text Available Two classes of ER stress sensors are known in plants, membrane associated bZIP transcription factors and RNA splicing factors. ER stress occurs under adverse environmental conditions and results from the accumulation of misfolded or unfolded proteins in the ER lumen. One of the membrane-associated transcription factors activated by heat and ER stress agents is bZIP28. In its inactive form, bZIP28 is a type II protein with a single pass transmembrane domain, residing in the ER. bZIP28’s N-terminus, containing a transcriptional activation domain, is oriented towards the cytoplasm and its C-terminal tail is inserted into the ER lumen. In response to stress, bZIP28 exits the ER and moves to the Golgi where it is proteolytically processed, liberating its cytosolic component which relocates to the nucleus to upregulate stress-response genes. bZIP28 is thought to sense stress through its interaction with the major ER chaperone, BIP. BiP binds to bZIP28’s lumenal domain under unstressed conditions and retains it in the ER. BIP binds to the intrinsically disordered regions on bZIP28’s lumen-facing tail. A truncated form of bZIP28, without its C-terminal tail is not retained in the ER but migrates constitutively to the nucleus. Upon stress, BiP releases bZIP28 allowing it to exit the ER. One model to account for the release of bZIP28 by BiP is that BiP is competed away from bZIP28 by the accumulation of misfolded proteins in the ER. However, other forces such as changes in energy charge levels, redox conditions or interaction with DNAJ proteins may also promote release of bZIP28 from BiP. Movement of bZIP28 from the ER to the Golgi is assisted by the interaction of elements of the COPII machinery with the cytoplasmic domain of bZIP28. Thus, the mobilization of bZIP28 in response to stress involves the dissociation of factors that retain it in the ER and the association of factors that mediate its further organelle-to-organelle movement.

  15. The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression

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    Fei eZhou

    2015-04-01

    Full Text Available The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA, we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD and constant dark (DD conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant.

  16. The bZIP transcription factor HY5 interacts with the promoter of the monoterpene synthase gene QH6 in modulating its rhythmic expression.

    Science.gov (United States)

    Zhou, Fei; Sun, Tian-Hu; Zhao, Lei; Pan, Xi-Wu; Lu, Shan

    2015-01-01

    The Artemisia annua L. β-pinene synthase QH6 was previously determined to be circadian-regulated at the transcriptional level, showing a rhythmic fluctuation of steady-state transcript abundances. Here we isolated both the genomic sequence and upstream promoter region of QH6. Different regulatory elements, such as G-box (TGACACGTGGCA, -421 bp from the translation initiation site) which might have effects on rhythmic gene expression, were found. Using the yeast one-hybrid and electrophoretic mobility shift assay (EMSA), we confirmed that the bZIP transcription factor HY5 binds to this motif of QH6. Studies with promoter truncations before and after this motif suggested that this G-box was important for the diurnal fluctuation of the transgenic β-glucuronidase gene (GUS) transcript abundance in Arabidopsis thaliana. GUS gene driven by the promoter region immediately after G-box showed an arrhythmic expression in both light/dark (LD) and constant dark (DD) conditions, whereas the control with G-box retained its fluctuation in both LD and DD. We further transformed A. thaliana with the luciferase gene (LUC) driven by an 1400 bp fragment upstream QH6 with its G-box intact or mutated, respectively. The luciferase activity assay showed that a peak in the early morning disappeared in the mutant. Gene expression analysis also demonstrated that the rhythmic expression of LUC was abolished in the hy5-1 mutant. PMID:25983739

  17. Orphan nuclear receptor Errγ induces C-reactive protein gene expression through induction of ER-bound Bzip transmembrane transcription factor CREBH.

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    Jagannath Misra

    Full Text Available The orphan nuclear receptor estrogen-related receptor-γ (ERRγ is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER stress response. cAMP responsive element-binding protein H (CREBH is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP, whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions.

  18. The Clock Protein CCA1 and the bZIP Transcription Factor HY5 Physically Interact to Regulate Gene Expression in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Christos Andronis; Simon Barak; Stephen M.Knowles; Shoji Sugano; Elaine M.Tobin

    2008-01-01

    The circadian clock regulates the expression of an array of Arabidopsis genes such as those encoding the LIGHT-HARVESTING CHLOROPHYLL A/B (Lhcb) proteins. We have previously studied the promoters of two of these Arabidopsis genes-Lhcb1*1 and Lhcb1*3-and identified a sequence that binds the clock protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). This sequence, designated CCAl-binding site (CBS), is necessary for phytochrome and circadian responsiveness of these genes. In close proximity to this sequence, there exists a G-box core element that has been shown to bind the bZIP transcription factor HY5 in other light-regulated plant promoters. In the present study, we examined the importance of the interaction of transcription factors binding the CBS and the G-box core element in the control of normal circadian rhythmic expression of Lhcb genes. Our results show that HY5 is able to specifically bind the G-box element in the Lhcb promoters and that CCA1 can alter the binding activity of HY5. We further show that CCA1 and HY5 can physically interact and that they can act synergistically on transcription in a yeast reporter gene assay. An absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression but does not affect the circadian expression of CATALASE3 (CAT3), whose promoter lacks a G-box element. Our results suggest that interaction of the HY5 and CCA1 proteins on Lhcb promoters is necessary for normal circadian expression of the Lhcb genes.

  19. The lumen-facing domain is important for the biological function and organelle-to-organelle movement of bZIP28 during ER stress in Arabidopsis.

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    Sun, Le; Lu, Sun-Jie; Zhang, Shuang-Shuang; Zhou, Shun-Fan; Sun, Ling; Liu, Jian-Xiang

    2013-09-01

    The membrane-associated transcription factor, bZIP28, is relocated from the endoplasmic reticulum (ER) to the Golgi and proteolytically released from the membrane mediated by two proteases, S1P and S2P, in response to ER stress in Arabidopsis. The activated N-terminal domain recruits nuclear factor Y (NF-Y) subunits in the nucleus to regulate ER stress downstream genes. Little is known about the functions of the bZIP28 C-terminal lumen-facing domain. Here, we provide novel insights into how the ER lumen-facing domain affects the biological function and organelle-to-organelle movement of bZIP28 in the ER stress response. First, we demonstrated the functional redundancy of bZIP28 and bZIP60 by generation and analysis of the bZIP28 and bZIP60 double mutant zip28zip60. Subsequent genetic complementation experiments in zip28zip60 background with deletions on bZIP28 lumen-facing domain highlighted the importance of lumen-facing domain for its in vivo function of bZIP28 in the ER stress response. The protein subcellular localization and Western blotting results further revealed that the bZIP28 lumen-facing domain contains ER retention signal which is important for the proteolytic activation of bZIP28. Thus, the bZIP28 lumen-facing C-terminus plays important roles in the ER-to-Golgi movement of bZIP28, which may contribute to the sensing of the ER stress.

  20. Feedback Regulation of ABA Signaling and Biosynthesis by a bZIP Transcription Factor Targets Drought-Resistance-Related Genes.

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    Zong, Wei; Tang, Ning; Yang, Jun; Peng, Lei; Ma, Siqi; Xu, Yan; Li, Guoliang; Xiong, Lizhong

    2016-08-01

    The OsbZIP23 transcription factor has been characterized for its essential role in drought resistance in rice (Oryza sativa), but the mechanism is unknown. In this study, we first investigated the transcriptional activation of OsbZIP23. A homolog of SnRK2 protein kinase (SAPK2) was found to interact with and phosphorylate OsbZIP23 for its transcriptional activation. SAPK2 also interacted with OsPP2C49, an ABI1 homolog, which deactivated the SAPK2 to inhibit the transcriptional activation activity of OsbZIP23. Next, we performed genome-wide identification of OsbZIP23 targets by immunoprecipitation sequencing and RNA sequencing analyses in the OsbZIP23-overexpression, osbzip23 mutant, and wild-type rice under normal and drought stress conditions. OsbZIP23 directly regulates a large number of reported genes that function in stress response, hormone signaling, and developmental processes. Among these targets, we found that OsbZIP23 could positively regulate OsPP2C49, and overexpression of OsPP2C49 in rice resulted in significantly decreased sensitivity of the abscisic acid (ABA) response and rapid dehydration. Moreover, OsNCED4 (9-cis-epoxycarotenoid dioxygenase4), a key gene in ABA biosynthesis, was also positively regulated by OsbZIP23. Together, our results suggest that OsbZIP23 acts as a central regulator in ABA signaling and biosynthesis, and drought resistance in rice. PMID:27325665

  1. Expression of a protein involved in bone resorption, Dkk1, is activated by HTLV-1 bZIP factor through its activation domain

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    Mesnard Jean-Michel

    2010-07-01

    Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 is the etiologic agent of adult T-cell leukemia, a malignancy characterized by uncontrolled proliferation of virally-infected CD4+ T-cells. Hypercalcemia and bone lesions due to osteoclast-mediated bone resorption are frequently associated with more aggressive forms of the disease. The HTLV-1 provirus contains a unique antisense gene that expresses HTLV-1 basic leucine zipper (bZIP factor (HBZ. HBZ is localized to the nucleus where it regulates levels of transcription by binding to certain cellular transcriptional regulators. Among its protein targets, HBZ forms a stable complex with the homologous cellular coactivators, p300 and CBP, which is modulated through two N-terminal LXXLL motifs in the viral protein and the conserved KIX domain in the coactivators. Results To determine the effects of these interactions on transcription, we performed a preliminary microarray analysis, comparing levels of gene expression in cells with wild-type HBZ versus cells with HBZ mutated in its LXXLL motifs. DKK1, which encodes the secreted Wnt signaling inhibitor, Dickkopf-1 (Dkk1, was confirmed to be transcriptionally activated by HBZ, but not its mutant. Dkk1 plays a major role in the development of bone lesions caused by multiple myeloma. In parallel with the initial findings, activation of Dkk1 expression by HBZ was abrogated by siRNA-mediated knockdown of p300/CBP or by a truncated form of p300 containing the KIX domain. Among HTLV-1-infected T-cell lines tested, the detection of Dkk1 mRNA partially correlated with a threshold level of HBZ mRNA. In addition, an uninfected and an HTLV-1-infected T-cell line transfected with an HBZ expression vector exhibited de novo and increased DKK1 transcription, respectively. In contrast to HBZ, The HTLV-1 Tax protein repressed Dkk1 expression. Conclusions These data indicate that HBZ activates Dkk1 expression through its interaction with p300/CBP. However

  2. The AP-1 transcription factor homolog Pf-AP-1 activates transcription of multiple biomineral proteins and potentially participates in Pinctada fucata biomineralization

    OpenAIRE

    Xiangnan Zheng; Minzhang Cheng; Liang Xiang; Jian Liang; Liping Xie; Rongqing Zhang

    2015-01-01

    Activator protein-1 (AP-1) is an important bZIP transcription factor that regulates a series of physiological processes by specifically activating transcription of several genes, and one of its well-chartered functions in mammals is participating in bone mineralization. We isolated and cloned the complete cDNA of a Jun/AP-1 homolog from Pinctada fucata and called it Pf-AP-1. Pf-AP-1 had a highly conserved bZIP region and phosphorylation sites compared with those from mammals. A tissue distrib...

  3. Differential expression of four soybean bZIP genes during Phakopsora pachyrhizi infection.

    Science.gov (United States)

    Alves, Murilo S; Soares, Zamira G; Vidigal, Pedro M P; Barros, Everaldo G; Poddanosqui, Adriana M P; Aoyagi, Luciano N; Abdelnoor, Ricardo V; Marcelino-Guimarães, Francismar C; Fietto, Luciano G

    2015-11-01

    Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is one of most important diseases in the soybean (Glycine max (L.) Merr.) agribusiness. The identification and characterization of genes related to plant defense responses to fungal infection are essential to develop ASR-resistant plants. In this work, we describe four soybean genes, GmbZIP62, GmbZIP105, GmbZIPE1, and GmbZIPE2, which encode transcription factors containing a basic leucine zipper (bZIP) domain from two divergent classes, and that are responsive to P. pachyrhizi infection. Molecular phylogenetic analyses demonstrated that these genes encode proteins similar to bZIP factors responsive to pathogens. Yeast transactivation assays showed that only GmbZIP62 has strong transactivation activity in yeast. In addition, three of the bZIP transcription factors analyzed were also differentially expressed by plant defense hormones, and all were differentially expressed by fungal attack, indicating that these proteins might participate in response to ASR infection. The results suggested that these bZIP proteins are part of the plant defense response to P. pachyrhizi infection, by regulating the gene expression related to ASR infection responses. These bZIP genes are potential targets to obtain new soybean genotypes resistant to ASR.

  4. Sucrose regulated translational control of bZip genes in Arabidopsis thaliana

    NARCIS (Netherlands)

    Rahmani, F.

    2007-01-01

    Sucrose can translationally regulate the expression of bZIP11 and four other S-class bZip transcription factors in Arabidopsis thaliana. Sequence encoding 28 amino acids (SC-peptide) in the leader of the bZIP11 is sufficient to mediate sucrose induced translational control. A model proposes that suc

  5. The Role of Plant bZIP Transcription Factors in Abiotic Stress%植物bZIP转录因子在非生物胁迫中的作用

    Institute of Scientific and Technical Information of China (English)

    冉静; 邹杰; 刘爱玲; 陈信波

    2012-01-01

    The abiotic stress factors, drought, salt and extreme temperature, have seriously impact on plant growth. The basic leucine zipper (bZIP) transcription factors, which were identified in various plants, have been proved to be involved in responding process to various abiotic stresses. The role of plant bZIP transcription factors in the response to abiotic stress and its latest advances were reviewed.%非生物胁迫因子如干旱、高温、低温和盐碱等严重影响着植物的生长发育,碱性亮氨酸拉链( bZIP)类转录因子与非生物逆境胁迫响应有密切关联.综述了植物bZIP类转录因子在应答非生物逆境胁迫中的作用及研究进展.

  6. Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

    Directory of Open Access Journals (Sweden)

    Hiu-Kwan Chow

    Full Text Available Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim protein Arnt fused to the leucine zipper (LZ dimerization domain from bZIP (basic region-leucine zipper protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H, transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed, as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d 148.9 nM and 40.2 nM, respectively, but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa. Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

  7. Bioinformatic cis-element analyses performed in Arabidopsis and rice disclose bZIP- and MYB-related binding sites as potential AuxRE-coupling elements in auxin-mediated transcription

    Directory of Open Access Journals (Sweden)

    Berendzen Kenneth W

    2012-08-01

    Full Text Available Abstract Background In higher plants, a diverse array of developmental and growth-related processes is regulated by the plant hormone auxin. Recent publications have proposed that besides the well-characterized Auxin Response Factors (ARFs that bind Auxin Response Elements (AuxREs, also members of the bZIP- and MYB-transcription factor (TF families participate in transcriptional control of auxin-regulated genes via bZIP Response Elements (ZREs or Myb Response Elements (MREs, respectively. Results Applying a novel bioinformatic algorithm, we demonstrate on a genome-wide scale that singular motifs or composite modules of AuxREs, ZREs, MREs but also of MYC2 related elements are significantly enriched in promoters of auxin-inducible genes. Despite considerable, species-specific differences in the genome structure in terms of the GC content, this enrichment is generally conserved in dicot (Arabidopsis thaliana and monocot (Oryza sativa model plants. Moreover, an enrichment of defined composite modules has been observed in selected auxin-related gene families. Consistently, a bipartite module, which encompasses a bZIP-associated G-box Related Element (GRE and an AuxRE motif, has been found to be highly enriched. Making use of transient reporter studies in protoplasts, these findings were experimentally confirmed, demonstrating that GREs functionally interact with AuxREs in regulating auxin-mediated transcription. Conclusions Using genome-wide bioinformatic analyses, evolutionary conserved motifs have been defined which potentially function as AuxRE-dependent coupling elements to establish auxin-specific expression patterns. Based on these findings, experimental approaches can be designed to broaden our understanding of combinatorial, auxin-controlled gene regulation.

  8. Rice bZIP protein, REB, interacts with GCN4 motif in promoter of Waxy gene

    Institute of Scientific and Technical Information of China (English)

    CHENG; Shijun; (程世军); WANG; Zongyang(王宗阳); HONG; Mengmin(洪孟民)

    2002-01-01

    A bifactorial endosperm box (EB), which contains an endosperm motif (EM) and a GCN4 motif, was found in rice Wx promoter. EB was found in 5′ upstream region of many seed storage protein genes accounting for these genes expression exclusive in endosperm among various cereals. Many reports demonstrated that the bZIP transcription activators isolated from wheat, barley and maize, etc. regulate the gene expression through binding to the GCN4 motif. In this research, we showed that GCN4 sequence could be recognized by nuclear proteins extracted from immature rice seeds. Furthermore, a rice bZIP protein, REB was isolated by using PCR method and REB fusion protein was expressed in E. coli. The results of gel shift analysis showed that REB could recognize and bind to the GCN4 motif in the Wx gene in addition to binding to the target sequence in the promoter of α-globulin.

  9. AtMyb7, a subgroup 4 R2R3 Myb, negatively regulates ABA-induced inhibition of seed germination by blocking the expression of the bZIP transcription factor ABI5

    KAUST Repository

    Kim, Junhyeok

    2014-08-27

    Various Myb proteins have been shown to play crucial roles in plants, including primary and secondary metabolism, determination of cell fate and identity, regulation of development and involvement in responses to biotic and abiotic stresses. The 126 R2R3 Myb proteins (with two Myb repeats) have been found in Arabidopsis; however, the functions of most of these proteins remain to be fully elucidated. In the present study, we characterized the function of AtMyb7 using molecular biological and genetic analyses. We used qRT-PCR to determine the levels of stress-response gene transcripts in wild-type and atmyb7 plants. We showed that ArabidopsisAtMyb7 plays a critical role in seed germination. Under abscisic acid (ABA) and high-salt stress conditions, atmyb7 plants showed a lower germination rate than did wild-type plants. Furthermore, AtMyb7 promoter:GUS seeds exhibited different expression patterns in response to variations in the seed imbibition period. AtMyb7 negatively controls the expression of the gene encoding bZIP transcription factor, ABI5, which is a key transcription factor in ABA signalling and serves as a crucial regulator of germination inhibition in Arabidopsis. © 2014 John Wiley & Sons Ltd.

  10. IRE1/bZIP60-mediated unfolded protein response plays distinct roles in plant immunity and abiotic stress responses.

    Directory of Open Access Journals (Sweden)

    Adrian A Moreno

    Full Text Available Endoplasmic reticulum (ER-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR, is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA. However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR, whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent

  11. The CREB Transcription Factor Controls Transcriptional Activity of the Human RIC8B Gene.

    Science.gov (United States)

    Maureira, Alejandro; Sánchez, Rodolfo; Valenzuela, Nicole; Torrejón, Marcela; Hinrichs, María V; Olate, Juan; Gutiérrez, José L

    2016-08-01

    Proper regulation of gene expression is essential for normal development, cellular growth, and differentiation. Differential expression profiles of mRNA coding for vertebrate Ric-8B during embryo and adult stages have been observed. In addition, Ric-8B is expressed in few cerebral nuclei subareas. These facts point to a dynamic control of RIC8B gene expression. In order to understand the transcriptional regulation of this gene, we searched for cis-elements in the sequence of the human RIC8B promoter region, identifying binding sites for the basic/leucine zipper (bZip) CREB transcription factor family (CRE sites) and C/EBP transcription factor family (C/EBP sites). CRE sites were found clustered near the transcription start site, while the C/EBP sites were found clustered at around 300 bp upstream the CRE sites. Here, we demonstrate the ability of CREB1 and C/EBPβ to bind their respective elements identified in the RIC8B promoter. Comparative protein-DNA interaction analyses revealed only the proximal elements as high affinity sites for CREB1 and only the distal elements as high affinity sites for C/EBPβ. Chromatin immunoprecipitation analyses, carried out using a human neuroblastoma cell line, confirmed the preferential association of CREB to the proximal region of the RIC8B promoter. By performing luciferase reporter assays, we found the CRE sites as the most relevant elements for its transcriptional activity. Taken together, these data show the existence of functional CREB and C/EBP binding sites in the human RIC8B gene promoter, a particular distribution of these sites and demonstrate a relevant role of CREB in stimulating transcriptional activity of this gene. J. Cell. Biochem. 117: 1797-1805, 2016. © 2016 Wiley Periodicals, Inc. PMID:26729411

  12. Frequency-Dependent Regulation of Follicle-Stimulating Hormone β by Pulsatile Gonadotropin-Releasing Hormone Is Mediated by Functional Antagonism of bZIP Transcription Factors ▿

    Science.gov (United States)

    Ciccone, Nick A.; Xu, Shuyun; Lacza, Charlemagne T.; Carroll, Rona S.; Kaiser, Ursula B.

    2010-01-01

    Oscillatory synthesis and secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), under the control of pulsatile hypothalamic gonadotropin-releasing hormone (GnRH), is essential for normal reproductive development and fertility. The molecular mechanisms by which various patterns of pulsatile GnRH regulate gonadotrope responsiveness remain poorly understood. In contrast to the α and LHβ subunit genes, FSHβ subunit transcription is preferentially stimulated at low rather than high frequencies of pulsatile GnRH. In this study, mutation of a cyclic AMP response element (CRE) within the FSHβ promoter resulted in the loss of preferential GnRH stimulation at low pulse frequencies. We hypothesized that high GnRH pulse frequencies might stimulate a transcriptional repressor(s) to attenuate the action of CRE binding protein (CREB) and show that inducible cAMP early repressor (ICER) fulfills such a role. ICER was not detected under basal conditions, but pulsatile GnRH stimulated ICER to a greater extent at high than at low pulse frequencies. ICER binds to the FSHβ CRE site to reduce CREB occupation and abrogates both maximal GnRH stimulation and GnRH pulse frequency-dependent effects on FSHβ transcription. These data suggest that ICER production antagonizes the stimulatory action of CREB to attenuate FSHβ transcription at high GnRH pulse frequencies, thereby playing a critical role in regulating cyclic reproductive function. PMID:20008557

  13. Structural basis of transcription activation.

    Science.gov (United States)

    Feng, Yu; Zhang, Yu; Ebright, Richard H

    2016-06-10

    Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme. PMID:27284196

  14. Genome-wide analyses of the bZIP family reveal their involvement in the development, ripening and abiotic stress response in banana.

    Science.gov (United States)

    Hu, Wei; Wang, Lianzhe; Tie, Weiwei; Yan, Yan; Ding, Zehong; Liu, Juhua; Li, Meiying; Peng, Ming; Xu, Biyu; Jin, Zhiqiang

    2016-01-01

    The leucine zipper (bZIP) transcription factors play important roles in multiple biological processes. However, less information is available regarding the bZIP family in the important fruit crop banana. In this study, 121 bZIP transcription factor genes were identified in the banana genome. Phylogenetic analysis showed that MabZIPs were classified into 11 subfamilies. The majority of MabZIP genes in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis of two banana genotypes revealed the differential expression patterns of MabZIP genes in different organs, in various stages of fruit development and ripening, and in responses to abiotic stresses, including drought, cold, and salt. Interaction networks and co-expression assays showed that group A MabZIP-mediated networks participated in various stress signaling, which was strongly activated in Musa ABB Pisang Awak. This study provided new insights into the complicated transcriptional control of MabZIP genes and provided robust tissue-specific, development-dependent, and abiotic stress-responsive candidate MabZIP genes for potential applications in the genetic improvement of banana cultivars. PMID:27445085

  15. Characterization of a bZIP gene highly expressed during ripening of the peach fruit.

    Science.gov (United States)

    Lovisetto, Alessandro; Guzzo, Flavia; Tadiello, Alice; Confortin, Enrico; Pavanello, Anna; Botton, Alessandro; Casadoro, Giorgio

    2013-09-01

    A ripening specific bZIP gene of peach was studied by ectopically expressing it in tomato. Two lines, with either a mild or a strong phenotype, respectively, were analyzed in detail. Transgenic fruit morphology was normal, yet the time spent to proceed through the various ripening stages was longer compared to wild type. In agreement with this finding the transgenic berries produced less ethylene, and also had a modified expression of some ripening-related genes that was particularly evident in berries with a strong phenotype. In particular, in the latter fruits polygalacturonase and lipoxygenase genes, but also genes coding for transcription factors (TFs) important for tomato ripening (i.e. TAGL1, CNR, APETALA2a, NOR) did not show the expected decreased expression in the red berries. As regards the RIN gene, its expression continued to increase in both mild and strong lines, and this is in agreement with the dilated ripening times. Interestingly, a metabolomic analysis of berries at various stages of ripening showed that the longer time spent by the transgenic berries to proceed from a stage to another was not due to a slackened metabolism. In fact, the differences in amount of stage-specific marker metabolites indicated that the transgenic berries had a very active metabolism. Therefore, the dilated ripening and the enhanced metabolism of the berries over-expressing the bZIP gene suggest that such gene might regulate ripening by acting as a pacemaker for some of the ripening metabolic pathways.

  16. Identification and characterization a novel transcription factor activator protein-1 in the sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Yang, Limeng; Li, Chenghua; Chang, Yaqing; Gao, Yinxue; Wang, Yi; Wei, Jing; Song, Jian; Sun, Ping

    2015-08-01

    The transcription factor activator protein-1 (AP-1) is an important gene expression regulator with typical Jun and region-leucine zipper (bZIP) domains and can respond to a plethora of physiological and pathological stimulus. In this study, we identified a novel AP-1 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjAP-1). The full-length of AjAP-1 was of 2944 bp including a 5' untranslated region (UTR) of 201 bp, a 3' UTR of 1753 bp and a putative open reading frame of 990 bp encoding a polypeptide of 329 amino acid residues. Two representative domains of Jun and bZIP as well as two nuclear localization signals (NLSs) were also detected in deduced amino acid of AjAP-1. Spatial distribution expression indicated that AjAP-1 was ubiquitously expressed in all examined tissues with predominant expression in the body wall, moderate in the tube feet, respiratory tree and colemocytes and slightly weak in the intestine and longitudinal muscle. Time-course expression analysis in intestine and coelomocytes revealed that AjAP-1 both reached its peak expression at 4 h after Vibrio splendidus challenge with a 2.6 and 8.2-fold increase compared to their control groups, respectively. Taken together, all these results suggested that AjAP-1 was a novel immune factor and might be involved in the processes of anti-bacteria response in sea cucumber. PMID:26093208

  17. Analysis of transcriptional activities of the Meq proteins present in highly virulent Marek's disease virus strains, RB1B and Md5.

    Science.gov (United States)

    Murata, Shiro; Okada, Tsukasa; Kano, Rika; Hayashi, Yuko; Hashiguchi, Tomoyuki; Onuma, Misao; Konnai, Satoru; Ohashi, Kazuhiko

    2011-08-01

    Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens. Recent field isolates of MDV have tended to exhibit increasing virulence, and MDV strains are currently classified into four categories based on their relative virulence. Meq, a putative MDV oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. MDV isolates display distinct diversity and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and subsequently, to observed increases in MDV oncogenicity. In this study, we introduced mutations into the meq gene and used dual luciferase reporter assays to analyze the transcriptional activities of the resulting Meq proteins to determine whether distinct mutations in Meq could be responsible for differences in transcriptional activity among MDV strains. A proline-to-alanine substitution at position 217, the second position of one of the proline direct repeats in the transactivation domain, enhanced the transactivation activity of Meq. In addition, we found that two substitutions at positions 283 and 320 affected transactivation activity. These results suggest that the distinct diversity of and point mutations in the Meq proteins are responsible for differences in transactivation activity among MDV strains.

  18. Activation of TORC1 transcriptional coactivator through MEKK1-induced phosphorylation.

    Science.gov (United States)

    Siu, Yeung-Tung; Ching, Yick-Pang; Jin, Dong-Yan

    2008-11-01

    CREB is a prototypic bZIP transcription factor and a master regulator of glucose metabolism, synaptic plasticity, cell growth, apoptosis, and tumorigenesis. Transducers of regulated CREB activity (TORCs) are essential transcriptional coactivators of CREB and an important point of regulation on which various signals converge. In this study, we report on the activation of TORC1 through MEKK1-mediated phosphorylation. MEKK1 potently activated TORC1, and this activation was independent of downstream effectors MEK1/MEK2, ERK2, JNK, p38, protein kinase A, and calcineurin. MEKK1 induced phosphorylation of TORC1 both in vivo and in vitro. Expression of the catalytic domain of MEKK1 alone in cultured mammalian cells sufficiently caused phosphorylation and subsequent activation of TORC1. MEKK1 physically interacted with TORC1 and stimulated its nuclear translocation. An activation domain responsive to MEKK1 stimulation was mapped to amino acids 431-650 of TORC1. As a physiological activator of CREB, interleukin 1alpha triggered MEKK1-dependent phosphorylation of TORC1 and its consequent recruitment to the cAMP response elements in the interleukin 8 promoter. Taken together, our findings suggest a new mechanism for regulated activation of TORC1 transcriptional coactivator and CREB signaling.

  19. The Elucidation of the Interactome of 16 Arabidopsis bZIP Factors Reveals Three Independent Functional Networks

    Science.gov (United States)

    Llorca, Carles Marco; Berendzen, Kenneth Wayne; Malik, Waqas Ahmed; Mahn, Stefan; Piepho, Hans-Peter; Zentgraf, Ulrike

    2015-01-01

    The function of the bZIP transcription factors is strictly dependent on their ability to dimerize. Heterodimerization has proven to be highly specific and is postulated to operate as a combinatorial mechanism allowing the generation of a large variety of dimers with unique qualities by specifically combining a small set of monomers; an assumption that has not yet been tested systematically. Here, the interaction pattern and the transactivation properties of 16 Arabidopsis thaliana bZIPs are examined in transiently transformed Arabidopsis protoplasts to deliver a perspective on the relationship between bZIP dimerization and function. An interaction matrix of bZIPs belonging to the C, G, H, and S1 bZIP groups was resolved by Bimolecular Fluorescent Complementation (BiFC) coupled to quantitative flow cytometric analysis, while an extensive GUS reporter gene assay was carried out to determine the effect of different bZIP pairs on the expression of four different known bZIP-targeted promoters. Statistical data treatment and complementary bioinformatic analysis were performed to substantiate the biological findings. According to these results, the 16 bZIPs interact in three isolated networks, within which their members dimerize non-specifically and exhibit a significant level of functional redundancy. A coherent explanation for these results is supported by in silico analysis of differences in the length, structure and composition of their leucine zippers and appears to explain their dimerization specificity and dynamics observed in vivo quite well. A model in which the bZIP networks act as functional units is proposed. PMID:26452049

  20. Intrinsic transcript cleavage activity of RNA polymerase.

    OpenAIRE

    Orlova, M; Newlands, J; Das, A; Goldfarb, A; Borukhov, S

    1995-01-01

    The GreA and GreB transcript cleavage factors of Escherichia coli suppress elongation arrest and may have a proofreading role in transcription. With the use of E. coli greA-greB- mutant, RNA polymerase is demonstrated to possess substantial intrinsic transcript cleavage activity. Mildly alkaline pH mimics the effect of the Gre proteins by inducing transcript cleavage in ternary complexes and antagonizing elongation arrest through a cleavage-and-restart reaction. Thus, transcript cleavage cons...

  1. The leucine zipper domains of the transcription factors GCN4 and c-Jun have ribonuclease activity.

    Directory of Open Access Journals (Sweden)

    Yaroslav Nikolaev

    Full Text Available Basic-region leucine zipper (bZIP proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3',5'-phosphodiester bonds with formation of 2',3'-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins. If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides.

  2. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site...

  3. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    Science.gov (United States)

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-01

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.

  4. Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

    Science.gov (United States)

    Hino, Kenta; Saito, Atsushi; Kido, Miori; Kanemoto, Soshi; Asada, Rie; Takai, Tomoko; Cui, Min; Cui, Xiang; Imaizumi, Kazunori

    2014-05-16

    The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis.

  5. Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

    Science.gov (United States)

    Hino, Kenta; Saito, Atsushi; Kido, Miori; Kanemoto, Soshi; Asada, Rie; Takai, Tomoko; Cui, Min; Cui, Xiang; Imaizumi, Kazunori

    2014-05-16

    The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis. PMID:24711445

  6. Chromatin structure near transcriptionally active genes

    International Nuclear Information System (INIS)

    Hypersensitive domains are the most prominent features of transcriptionally active chromatin. In the case of the β/sup A/-globin gene, it seems likely that two or more protein factors are capable of binding to the DNA so tightly that the nucleosome is prevented from binding. We have shown that nucleosomes, once bound in the assembly process in vitro, cannot be displaced. The interaction of the 5S gene transcription factor TFIIIA with its target DNA also is blocked by histones, and it has been suggested that the activation of the gene must occur during replication, before histones are reassembled on the DNA. We suppose that a similar mechanism may govern the binding of the hypersensitivity factors. It should be noted that nucleosomes are excluded not only from the sites to which the factors bind, but also from the regions between the two domains and at either side. 12 refs., 6 figs

  7. Transcriptional template activity of covalently modified DNA.

    Science.gov (United States)

    Tolwińska-Stańczyk, Z; Wilmańska, D; Studzian, K; Gniazdowski, M

    1997-03-01

    The transcriptional template activity of covalent modified DNA is compared. 8-Methoxypsoralen (MOP), 3,4'dimethyl-8-methoxypsoralen (DMMOP) and benzopsoralen (BP) forming with DNA covalent complexes upon UV irradiation and exhibiting preference to pyrimidines, mostly thymines, differ in their cross-linking potency. MOP and DMMOP form both monoadducts and diadducts while no cross-links are formed by BP. Nitracrine (NC) forms covalent complexes with DNA upon reductive activation with dithiothreitol exhibiting a preference to purines and low cross-linking potency. Semilogarithmic plots of the relative template activity against the number of the drugs molecules covalently bound per 10(3) DNA nucleotides fit to regression lines corresponding to one-hit inactivation characteristics. The number of drug molecules decreasing RNA synthesis to 37% differ from 0.25 to 1.26 depending on the template used and the base preference but no dependence on the cross-linking potency was found. PMID:9067423

  8. Genomic surveys and expression analysis of bZIP gene family in castor bean (Ricinus communis L.).

    Science.gov (United States)

    Jin, Zhengwei; Xu, Wei; Liu, Aizhong

    2014-02-01

    The basic leucine zipper (bZIP) transcription factors comprise a family of transcriptional regulators present extensively in plants, involved in regulating diverse biological processes such as flower and vascular development, seed maturation, stress signaling and pathogen defense. Castor bean (Ricinus communis L. Euphorbiaceae) is one of the most important non-edible oilseed crops and its seed oil is broadly used for industrial applications. We performed a comprehensive genome-wide identification and analysis of the bZIP transcription factors that exist in the castor bean genome in this study. In total, 49 RcbZIP transcription factors were identified, characterized and categorized into 11 groups (I-XI) based on their gene structure, DNA-binding sites, conserved motifs, and phylogenetic relationships. The dimerization properties of 49 RcbZIP proteins were predicted on the basis of the characteristic features in the leucine zipper. Global expression profiles of 49 RcbZIP genes among different tissues were examined using high-throughput sequencing of digital gene expression profiles, and resulted in diverse expression patterns that may provide basic information to further reveal the function of the 49 RcbZIP genes in castor bean. The results obtained from this study would provide valuable information in understanding the molecular basis of the RcbZIP transcription factor family and their potential function in regulating the growth and development, particularly in seed filling of castor bean.

  9. [The Effect of Transcription on Enhancer Activity in Drosophila melanogaster].

    Science.gov (United States)

    Erokhin, M M; Davydova, A I; Lomaev, D V; Georgiev, P G; Chetverina, D A

    2016-01-01

    In higher eukaryotes, the level of gene transcription is under the control of DNA regulatory elements, such as promoter, from which transcription is initiated with the participation of RNA polymerase II and general transcription factors, as well as the enhancer, which increase the rate of transcription with the involvement of activator proteins and cofactors. It was demonstrated that enhancers are often located in the transcribed regions of the genome. We showed earlier that transcription negatively affected the activity of enhancers in Drosophila in model transgenic systems. In this study, we tested the effect of the distance between the leading promoter, enhancer, and target promoter on the inhibitory effect of transcriptions of different strengths. It was demonstrated that the negative effect of transcription remained, but weakened with increased distance between the leading promoter and enhancer and with decreased distance between the enhancer and target promoter. Thus, transcription can modulate the activity of enhancers by controlling its maximum level.

  10. Human cytomegalovirus IE2 protein interacts with transcription activating factors

    Institute of Scientific and Technical Information of China (English)

    徐进平; 叶林柏

    2002-01-01

    The human cytomegalovirus (HCMV) IE86 Cdna was cloned into Pgex-2T and fusion protein GST-IE86 was expressed in E. Coli. SDS-PAGE and Western blot assay indicated that fusion protein GST-IE86 with molecular weight of 92 ku is soluble in the supernatant of cell lysate. Protein GST and fusion protein GST-IE86 were purified by affinity chromatography. The technology of co-separation and specific affinity chromatography was used to study the interactions of HCMV IE86 protein with some transcriptional regulatory proteins and transcriptional factors. The results indicated that IE86 interacts separately with transcriptional factor TFIIB and promoter DNA binding transcription trans-activating factors SP1, AP1 and AP2 to form a heterogenous protein complex. These transcriptional trans-activating factors, transcriptional factor and IE86 protein were adsorbed and retained in the affinity chromatography simultaneously. But IE86 protein could not interact with NF-Кb, suggesting that the function of IE86 protein that can interact with transcriptional factor and transcriptional trans-activating factors has no relevance to protein glycosylation. IE86 protein probably has two domains responsible for binding transcriptional trans-activating regulatory proteins and transcriptional factors respectively, thus activating the transcription of many genes. The interactions accelerated the assembly of the transcriptional initiation complexes.

  11. Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation.

    Science.gov (United States)

    Hao, Yu-Jun; Song, Qing-Xin; Chen, Hao-Wei; Zou, Hong-Feng; Wei, Wei; Kang, Xu-Sheng; Ma, Biao; Zhang, Wan-Ke; Zhang, Jin-Song; Chen, Shou-Yi

    2010-10-01

    Plant-specific transcription factor NAC proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. In the NAC family, some members function as transcription activators while others act as repressors. In the present study we found that though the full-length GmNAC20 from soybean did not have transcriptional activation activity, the carboxy-terminal activation domain of GmNAC20 had high transcriptional activation activity in the yeast assay system. Deletion experiments revealed an active repression domain with 35 amino acids, named NARD (NAC Repression Domain), in the d subdomain of NAC DNA-binding domain. NARD can reduce the transcriptional activation ability of diverse transcription factors when fused to either the amino-terminal or the carboxy-terminal of the transcription factors. NARD-like sequences are also present in other NAC family members and they are functional repression domain when fused to VP16 in plant protoplast assay system. Mutation analysis of conserved amino acid residues in NARD showed that the hydrophobic LVFY motif may partially contribute to the repression function. It is hypothesized that the interactions between the repression domain NARD and the carboxy-terminal activation domain may finally determine the ability of NAC family proteins to regulate downstream gene expressions.

  12. Zea mays Taxilin protein negatively regulates opaque-2 transcriptional activity by causing a change in its sub-cellular distribution.

    Directory of Open Access Journals (Sweden)

    Nan Zhang

    Full Text Available Zea mays (maize Opaque-2 (ZmO2 protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2 as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.

  13. 拟南芥bZIP1转录因子通过与ABRE元件结合调节ABA信号传导%Arabidopisis bZIP1 Transcription Factor Binding to the ABRE Cis-Element Regulates Abscisic Acid Signal Transduction

    Institute of Scientific and Technical Information of China (English)

    孙晓丽; 李勇; 才华; 柏锡; 纪巍; 季佐军; 朱延明

    2011-01-01

    Abscisic acid (ABA) is a phytohormone and mediates the response and adaptation of higher plants to various environmental stresses during vegetative growth.The basic leucine zipper (bZIP) transcription factors are also important regulators of plant development and abiotic resistance, acting through either ABA-dependent or ABA-independent pathways.In this study, we investigated and characterized the involvement of the AtbZIP1 gene in plant responsiveness to ABA.As confirmed by PCR and RT-PCR, AtbZIP1 has been silenced in mutant Arabidopsis ko-1 (SALK_059343) and ko-2 (SALK_069489C).The AtbZIP1 knockout plants demonstrated reduced sensitivity to ABA both at the seed germination and seedling stage with improvements in rates of germination, leaf opening/greening, and primary root length.In order to investigate whether the regulation of AtbZIP1-mediated ABA responsiveness depended on the ABA-responsive elements (ABRE), we expressed the AtbZIP1 HIS6 fusion protein in E.coli and found that the AtbZIP1 HIS6 specifically bound to the ABRE cis-elements.Semi-quantitive RT PCR showed that AtbZIP1 disruption altered expressions of some ABA responsive genes, such as NCED3, RD22, KIN1, and RD29A.Our results indicated that AtbZIP1 regulates abscisic acid signal transduction by binding to the ABREs and altered the expressions of the ABA responsive genes.%ABA作为一种重要的植物激素和生长凋节剂,介导了高等植物在营养生长阶段对各种外界环境的响应和适应.bZIP类转录因子可以通过ABA依赖途径和ABA非依赖途径调节植物的生长发育和对非生物胁迫的耐性.本研究通过AtbZIP1 T-DNA插入突变的拟南芥植株ko-1(SALK_059343)和ko-2(SALK_069489C)在ABA处理后的表型实验,验证了AtbZIP1参与ABA依赖的信号传导通路.采用"三引物法",分别在DNA水平和RNA水平通过PCR和RT-PCR验证了AtbZIP1基因在拟南芥突变体中的沉默效果.定量分析数据表明,在种子萌发阶段,经过0.6μmolL-1

  14. Enhancer-activated plasmid transcription complexes contain constrained supercoiling.

    OpenAIRE

    Bonilla, P J; Freytag, S O; Lutter, L C

    1991-01-01

    It has been proposed that transcriptionally active chromatin contains totally unconstrained supercoiling. The results of recent studies have raised the possibility that this topological state is the property of highly transcribed genes. Since the transcription rate of RNA polymerase II genes can be dramatically increased by the presence of an enhancer, we have determined if the transcription complex of an enhancer-activated plasmid contains totally unconstrained supercoils. Following transfec...

  15. Regulating expression of cell and tissue-specific genes by modifying transcription

    Energy Technology Data Exchange (ETDEWEB)

    Beachy, Roger N; Dai, Shunhong

    2010-06-14

    Transcriptional regulation is the primary step to control gene expression, therefore function. Such regulation is achieved primarily via a combination of the activities of the promoter cis regulatory DNA elements and trans regulatory proteins that function through binding to these DNA elements. Rice bZIP transcription factors RF2a, RF2b and RLP1 play key roles in regulating the activity of a vascular tissue specific promoter isolated from Rice Tungro Bacilliform Virus (RTBV), through their interactions with the Box II essential cis element located in the promoter (Dai et al., 2006., Dai et al., 2004., Yin et al., 1997). RF2a, RF2b and RLP1 possess multiple regulatory domains. Functional characterization reveals that those domains can activate or repress the activity of the RTBV promoter. It is equally as important to recognize that these proteins control plant development by regulating differentiation and/or function of the vascular tissues. Studies of transcriptional regulation of the RTBV promoter by this group of bZIP proteins will not only provide insights about gene expression in the vascular tissue, but also insights about general mechanisms of transcription activation and repression. The knowledge gained from this research will also enable us to develop a well-described set of tools that can be used to control expression of multiple genes in transgenic plants. We have proposed characterize the function domains of RF2a, RF2b and RLP1 and explore the biological function of the transcription repressor RLP1.

  16. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  17. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

    Nakatsubo, Takuya; Nishitani, Saori; Kikuchi, Yuko; Iida, Satoshi; Yamada, Kana; Tanaka, Aki; Ohkuma, Yoshiaki

    2014-10-01

    In eukaryotes, the Mediator complex is an essential transcriptional cofactor of RNA polymerase II (Pol II). In humans, it contains up to 30 subunits and consists of four modules: head, middle, tail, and CDK/Cyclin. One of the subunits, MED15, is located in the tail module, and was initially identified as Gal11 in budding yeast, where it plays an essential role in the transcriptional regulation of galactose metabolism with the potent transcriptional activator Gal4. For this reason, we investigated the function of the human MED15 subunit (hMED15) in transcriptional activation. First, we measured the effect of hMED15 knockdown on cell growth in HeLa cells. The growth rate was greatly reduced. By immunostaining, we observed the colocalization of hMED15 with the general transcription factors TFIIE and TFIIH in the nucleus. We measured the effects of siRNA-mediated knockdown of hMED15 on transcriptional activation using two different transcriptional activators, VP16 and SREBP1a. Treatment with siRNAs reduced transcriptional activation, and this reduction could be rescued by overexpression of HA/Flag-tagged, wild-type hMED15. To investigate hMED15 localization, we treated human MCF-7 cells with the MDM2 inhibitor Nutlin-3, thus inducing p21 transcription. We found that hMED15 localized to both the p53 binding site and the p21 promoter region, along with TFIIE and TFIIH. These results indicate that hMED15 promotes transcriptional activation.

  18. Activation of archaeal transcription mediated by recruitment of transcription factor B.

    Science.gov (United States)

    Ochs, Simon M; Thumann, Sybille; Richau, Renate; Weirauch, Matt T; Lowe, Todd M; Thomm, Michael; Hausner, Winfried

    2012-05-25

    Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested. PMID:22496454

  19. Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2.

    Science.gov (United States)

    Luo, Qianyi; Viste, Kristin; Urday-Zaa, Janny Concha; Senthil Kumar, Ganesan; Tsai, Wen-Wei; Talai, Afsaneh; Mayo, Kelly E; Montminy, Marc; Radhakrishnan, Ishwar

    2012-12-18

    Basic leucine zipper (bZip) transcription factors regulate cellular gene expression in response to a variety of extracellular signals and nutrient cues. Although the bZip domain is widely known to play significant roles in DNA binding and dimerization, recent studies point to an additional role for this motif in the recruitment of the transcriptional apparatus. For example, the cAMP response element binding protein (CREB)-regulated transcriptional coactivator (CRTC) family of transcriptional coactivators has been proposed to promote the expression of calcium and cAMP responsive genes, by binding to the CREB bZip in response to extracellular signals. Here we show that the CREB-binding domain (CBD) of CRTC2 folds into a single isolated 28-residue helix that seems to be critical for its interaction with the CREB bZip. The interaction is of micromolar affinity on palindromic and variant half-site cAMP response elements (CREs). The CBD and CREB assemble on the CRE with 2:2:1 stoichiometry, consistent with the presence of one CRTC binding site on each CREB monomer. Indeed, the CBD helix and the solvent-exposed residues in the dimeric CREB bZip coiled-coil form an extended protein-protein interface. Because mutation of relevant bZip residues in this interface disrupts the CRTC interaction without affecting DNA binding, our results illustrate that distinct DNA binding and transactivation functions are encoded within the structural constraints of a canonical bZip domain.

  20. Regulating expressin of cell and tissue-specific genes by modifying transcription

    Energy Technology Data Exchange (ETDEWEB)

    Beachy, R N; Dai, Shunhong

    2009-12-15

    Transcriptional regulation is the primary step to control gene expression, therefore function. Such regulation is achieved primarily via a combination of the activities of the promoter cis regulatory DNA elements and trans regulatory proteins that function through binding to these DNA elements. Our research supported by this program has led to the identification of rice bZIP transcription factors RF2a, RF2b and RLP1 that play key roles in regulating the activity of a vascular tissue specific promoter isolated from Rice Tungro Bacilliform Virus (RTBV) through their interactions with the Box II essential cis element located in the promoter. RF2a, RF2b and RLP1 possess multiple regulatory domains. Functional characterization reveals that those domains can activate or repress the activity of the RTBV promoter. Studies of transcriptional regulation of the RTBV promoter by this group of bZIP proteins not only provide insights about gene expression in the vascular tissue, but also insights about general mechanisms of transcription activation and repression. The knowledge gained from this research will also enable us to develop a well-described set of tools that can be used to control expression of multiple genes in transgenic plants and to improve biofuel feedstock.

  1. Transcriptional Activation of the Zygotic Genome in Drosophila.

    Science.gov (United States)

    Harrison, Melissa M; Eisen, Michael B

    2015-01-01

    During the first stages of metazoan development, the genomes of the highly specified sperm and egg must unite and be reprogrammed to allow for the generation of a new organism. This process is controlled by maternally deposited products. Initially, the zygotic genome is largely transcriptionally quiescent, and it is not until hours later that the zygotic genome takes control of development. The transcriptional activation of the zygotic genome is tightly coordinated with the degradation of the maternal products. Here, we review the current understanding of the processes that mediate the reprogramming of the early embryonic genome and facilitate transcriptional activation during the early stages of Drosophila development.

  2. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise; Snoek, Tim; Kildegaard, Kanchana Rueksomtawin;

    2016-01-01

    real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily......,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications....

  3. The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

    Science.gov (United States)

    Tiwari, Shiv B; Belachew, Alemu; Ma, Siu Fong; Young, Melinda; Ade, Jules; Shen, Yu; Marion, Colleen M; Holtan, Hans E; Bailey, Adina; Stone, Jeffrey K; Edwards, Leslie; Wallace, Andreah D; Canales, Roger D; Adam, Luc; Ratcliffe, Oliver J; Repetti, Peter P

    2012-06-01

    In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

  4. Heterogeneity of Calcium Channel/cAMP-Dependent Transcriptional Activation.

    Science.gov (United States)

    Kobrinsky, Evgeny

    2015-01-01

    The major function of the voltage-gated calcium channels is to provide the Ca(2+) flux into the cell. L-type voltage-gated calcium channels (Cav1) serve as voltage sensors that couple membrane depolarization to many intracellular processes. Electrical activity in excitable cells affects gene expression through signaling pathways involved in the excitation-transcription (E-T) coupling. E-T coupling starts with activation of the Cav1 channel and results in initiation of the cAMP-response element binding protein (CREB)-dependent transcription. In this review we discuss the new quantitative approaches to measuring E-T signaling events. We describe the use of wavelet transform to detect heterogeneity of transcriptional activation in nuclei. Furthermore, we discuss the properties of discovered microdomains of nuclear signaling associated with the E-T coupling and the basis of the frequency-dependent transcriptional regulation.

  5. Dataset of transcriptional landscape of B cell early activation

    Directory of Open Access Journals (Sweden)

    Alexander S. Garruss

    2015-09-01

    Full Text Available Signaling via B cell receptors (BCR and Toll-like receptors (TLRs result in activation of B cells with distinct physiological outcomes, but transcriptional regulatory mechanisms that drive activation and distinguish these pathways remain unknown. At early time points after BCR and TLR ligand exposure, 0.5 and 2 h, RNA-seq was performed allowing observations on rapid transcriptional changes. At 2 h, ChIP-seq was performed to allow observations on important regulatory mechanisms potentially driving transcriptional change. The dataset includes RNA-seq, ChIP-seq of control (Input, RNA Pol II, H3K4me3, H3K27me3, and a separate RNA-seq for miRNA expression, which can be found at Gene Expression Omnibus Dataset GSE61608. Here, we provide details on the experimental and analysis methods used to obtain and analyze this dataset and to examine the transcriptional landscape of B cell early activation.

  6. Potential Role of Activating Transcription Factor 5 during Osteogenesis

    Directory of Open Access Journals (Sweden)

    Luisa Vicari

    2016-01-01

    Full Text Available Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2, encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology.

  7. Fungal mediator tail subunits contain classical transcriptional activation domains.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

  8. First Exon Length Controls Active Chromatin Signatures and Transcription

    Directory of Open Access Journals (Sweden)

    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  9. Estrogen directly activates AID transcription and function

    OpenAIRE

    Pauklin, Siim; Sernández, Isora V.; Bachmann, Gudrun; Ramiro, Almudena R.; Petersen-Mahrt, Svend K.

    2009-01-01

    The immunological targets of estrogen at the molecular, humoral, and cellular level have been well documented, as has estrogen's role in establishing a gender bias in autoimmunity and cancer. During a healthy immune response, activation-induced deaminase (AID) deaminates cytosines at immunoglobulin (Ig) loci, initiating somatic hypermutation (SHM) and class switch recombination (CSR). Protein levels of nuclear AID are tightly controlled, as unregulated expression can lead to alterations in th...

  10. Lithium enhances CRTC oligomer formation and the interaction between the CREB coactivators CRTC and CBP--implications for CREB-dependent gene transcription.

    Science.gov (United States)

    Heinrich, Annette; von der Heyde, Anne Sophie; Böer, Ulrike; Phu, Do Thanh; Tzvetkov, Mladen; Oetjen, Elke

    2013-01-01

    Lithium salts are important drugs to treat bipolar disorder. Previous work showed that lithium by enforcing the interaction between the transcription factor CREB and its coactivator CRTC1 enhanced cAMP-stimulated CREB-dependent gene transcription. Both CREB and CRTC have been implicated in neuronal adaptation, which might underlie lithium's therapeutic action. In the present study the mechanisms of lithium action on cAMP-induced CREB-dependent gene transcription were further elucidated. Transient transfection assays revealed that all three CRTC isoforms conferred lithium responsiveness to CREB whereas their intrinsic transcriptional activities remained unchanged by lithium, suggesting a conformational change of CREB or CRTC by lithium. In in vitro protein-protein interaction assays lithium enhanced the interaction between CREB and both coactivators CRTC and CBP. Furthermore, lithium enforced the oligomerization of CRTC, a prerequisite for CREB interaction. For further evaluation it was investigated whether lithium competes with magnesium, which coordinates the conformation of the CREB basic region leucine zipper (bZip). Mutational analysis of the magnesium coordinating lysine-290 within the bZip, in vitro and intracellular interaction assays and luciferase reporter-gene assays revealed that the effect of lithium on the CREB-CRTC interaction or on the transcriptional activity, respectively, was not affected by the mutation, thus excluding a magnesium-lithium competition. However, the CREB-CRTC interaction was strongly increased in lysine-290-mutants thereby extending the CRTC-CREB interaction domain. Taken together the results exclude a competition between lithium and magnesium at the bZip, but suggest that lithium by enforcing the CRTC-oligomer formation and the interaction of CREB-CBP-CRTC enhances cAMP-induced CREB-dependent gene transcription.

  11. Transcriptional activity of transposable elements in coelacanth.

    Science.gov (United States)

    Forconi, Mariko; Chalopin, Domitille; Barucca, Marco; Biscotti, Maria Assunta; De Moro, Gianluca; Galiana, Delphine; Gerdol, Marco; Pallavicini, Alberto; Canapa, Adriana; Olmo, Ettore; Volff, Jean-Nicolas

    2014-09-01

    The morphological stasis of coelacanths has long suggested a slow evolutionary rate. General genomic stasis might also imply a decrease of transposable elements activity. To evaluate the potential activity of transposable elements (TEs) in "living fossil" species, transcriptomic data of Latimeria chalumnae and its Indonesian congener Latimeria menadoensis were compared through the RNA-sequencing mapping procedures in three different organs (liver, testis, and muscle). The analysis of coelacanth transcriptomes highlights a significant percentage of transcribed TEs in both species. Major contributors are LINE retrotransposons, especially from the CR1 family. Furthermore, some particular elements such as a LF-SINE and a LINE2 sequences seem to be more expressed than other elements. The amount of TEs expressed in testis suggests possible transposition burst in incoming generations. Moreover, significant amount of TEs in liver and muscle transcriptomes were also observed. Analyses of elements displaying marked organ-specific expression gave us the opportunity to highlight exaptation cases, that is, the recruitment of TEs as new cellular genes, but also to identify a new Latimeria-specific family of Short Interspersed Nuclear Elements called CoeG-SINEs. Overall, transcriptome results do not seem to be in line with a slow-evolving genome with poor TE activity.

  12. Trans-dominant inhibition of transcription activator LFB1.

    OpenAIRE

    Nicosia, A.; Tafi, R; Monaci, P

    1992-01-01

    Liver-enriched factor LFB1 (also named HNF1) is a dimeric transcription activator which is essential for the expression of many hepatocyte-specific genes. Here we demonstrate that LFB1 mutants in the POU A-like or in the homeo domains inhibit wild-type DNA binding by forming inactive heterodimeric complexes. Co-transfection of one of these mutants with wild-type LFB1 in HeLa cells eliminated LFB1 DNA binding and transcriptional activities through a trans-dominant mechanism. Expression of the ...

  13. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus.

    Science.gov (United States)

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-03-18

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer-promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them.

  14. Bidirectional Transcription Arises from Two Distinct Hubs of Transcription Factor Binding and Active Chromatin.

    Science.gov (United States)

    Scruggs, Benjamin S; Gilchrist, Daniel A; Nechaev, Sergei; Muse, Ginger W; Burkholder, Adam; Fargo, David C; Adelman, Karen

    2015-06-18

    Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the regulation or function of anti-sense promoters and the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNA genes. We find that coupled sense and anti-sense TSSs precisely define the boundaries of a nucleosome-depleted region (NDR) that is highly enriched in transcription factor (TF) motifs. Notably, as the distance between sense and anti-sense TSSs increases, so does the size of the NDR, the level of signal-dependent TF binding, and gene activation. We further discover a group of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Interestingly, this signature identifies divergent promoters that are activated during immune challenge. We propose that anti-sense promoters serve as platforms for TF binding and establishment of active chromatin to further regulate or enhance sense-strand mRNA expression.

  15. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

    Full Text Available BACKGROUND: The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. METHODOLOGY: The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE. RESULTS: AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. CONCLUSION: We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  16. The transcriptionally active regions in the genome of Bacillus subtilis

    DEFF Research Database (Denmark)

    Rasmussen, Simon; Nielsen, Henrik Bjørn; Jarmer, Hanne Østergaard

    2009-01-01

    The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome...

  17. HAT activity is essential for CBP-1-dependent transcription and differentiation in Caenorhabditis elegans

    OpenAIRE

    Victor, Martin; Bei, Yanxia; Gay, Frédérique; Calvo, Dominica; Mello, Craig; Shi, Yang

    2002-01-01

    The p300/CBP family of transcriptional coactivators possesses multiple functional domains, including a histone acetyltransferase (HAT) and several activation domains. A number of models have been proposed to account for their roles in transcriptional activation, including interactions with basal transcription machinery and chromatin remodeling. However, individual contributions of these domains to transcriptional activation and their significance in living organisms remain unclear. We address...

  18. Transcriptional Regulatory Circuits Controlling Brown Fat Development and Activation

    OpenAIRE

    Seale, Patrick

    2015-01-01

    Brown and beige adipose tissue is specialized for heat production and can be activated to reduce obesity and metabolic dysfunction in animals. Recent studies also have indicated that human brown fat activity levels correlate with leanness. This has revitalized interest in brown fat biology and has driven the discovery of many new regulators of brown fat development and function. This review summarizes recent advances in our understanding of the transcriptional mechanisms that control brown an...

  19. An upstream activation element exerting differential transcriptional activation on an archaeal promoter

    DEFF Research Database (Denmark)

    Peng, Nan; Xia, Qiu; Chen, Zhengjun;

    2009-01-01

    (UAS) ara-box activated the basal promoter by recruiting transcription factor B to its BRE. While this UAS ensured a general expression from an inactive or weak basal promoter in the presence of other tested carbon resources, it exhibited a strong arabinose-responsive transcriptional activation. To our...

  20. Assembly of a Notch transcriptional activation complex requires multimerization.

    Science.gov (United States)

    Vasquez-Del Carpio, Rodrigo; Kaplan, Fred M; Weaver, Kelly L; VanWye, Jeffrey D; Alves-Guerra, Marie-Clotilde; Robbins, David J; Capobianco, Anthony J

    2011-04-01

    Notch transmembrane receptors direct essential cellular processes, such as proliferation and differentiation, through direct cell-to-cell interactions. Inappropriate release of the intracellular domain of Notch (N(ICD)) from the plasma membrane results in the accumulation of deregulated nuclear N(ICD) that has been linked to human cancers, notably T-cell acute lymphoblastic leukemia (T-ALL). Nuclear N(ICD) forms a transcriptional activation complex by interacting with the coactivator protein Mastermind-like 1 and the DNA binding protein CSL (for CBF-1/Suppressor of Hairless/Lag-1) to regulate target gene expression. Although it is well understood that N(ICD) forms a transcriptional activation complex, little is known about how the complex is assembled. In this study, we demonstrate that N(ICD) multimerizes and that these multimers function as precursors for the stepwise assembly of the Notch activation complex. Importantly, we demonstrate that the assembly is mediated by N(ICD) multimers interacting with Skip and Mastermind. These interactions form a preactivation complex that is then resolved by CSL to form the Notch transcriptional activation complex on DNA.

  1. Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation.

    Science.gov (United States)

    Amelio, Antonio L; Caputi, Massimo; Conkright, Michael D

    2009-09-16

    The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. Here we show that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1-MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity.

  2. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

    Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

  3. Characterization of the transcriptional activation domains of human TEF3-1 (transcription enhancer factor 3 isoform 1).

    Science.gov (United States)

    Qiao, Cheng; Jiang, Yajie; Deng, Cuilan; Huang, Zebo; Teng, Kaixuan; Chen, Lan; Liu, Xin

    2015-03-01

    TEF3-1 (transcription enhancer factor 3 isoform 1) is a human transcriptional factor, which has a N-terminal TEA/ATTS domain supposedly for DNA binding and C-terminal PRD and STY domains for transcriptional activation. Taking advantage of the efficient reporter design of yeast two-hybrid system, we characterized the TEF3-1 domains in activating gene expression. Previously study usually mentioned that the C-terminal domain of TEF3-1 has the transcriptional activity, however, our data shows that the peptides TEF3-11-66 and TEF3-1197-434 functioned as two independent activation domains, suggesting that N-terminal domain of TEF3-1 also has transcriptional activation capacity. Additionally, more deletions of amino acids 197-434 showed that only the peptides TEF3-1197-265 contained the minimum sequences for the C-terminal transcriptional activation domain. The protein structure is predicted to contain a helix-turn-helix structure in TEF3-11-66 and four β sheets in TEF3-1197-265. Finally, after the truncated fragments of TEF3-1 were expressed in HUVEC cells, the whole TEF3-1 and the two activation domains could increase F-actin stress fiber, cell proliferation, migration and targeted gene expression. Further analysis and characterization of the activation domains in TEF3-1 may broaden our understanding of the gene involved in angiogenesis and other pathological processes.

  4. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

    Science.gov (United States)

    Naranjo, José R.; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M.; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C.; Arrabal, María D.; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-01-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  5. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

    Science.gov (United States)

    Naranjo, José R; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C; Arrabal, María D; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-02-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  6. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

    Science.gov (United States)

    Naranjo, José R; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C; Arrabal, María D; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-02-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

  7. Peroxisome proliferator-activated receptor gamma recruits the positive transcription elongation factor b complex to activate transcription and promote adipogenesis

    DEFF Research Database (Denmark)

    Iankova, Irena; Petersen, Rasmus K; Annicotte, Jean-Sébastien;

    2006-01-01

    in adipogenesis. In this study we show that the expression of the cdk9 p55 isoform is highly regulated during 3T3-L1 adipocyte differentiation at RNA and protein levels. Furthermore, cdk9, as well as cyclin T1 and cyclin T2, shows differences in nuclear localization at distinct stages of adipogenesis...... with and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master regulator of this process, on the promoter of PPARgamma target genes. PPARgamma-cdk9 interaction results in increased transcriptional activity of PPARgamma and therefore increased adipogenesis....

  8. Distinct regulatory mechanisms of eukaryotic transcriptional activation by SAGA and TFIID.

    Science.gov (United States)

    Bhaumik, Sukesh R

    2011-02-01

    A growing number of human diseases are linked to abnormal gene expression which is largely controlled at the level of transcriptional initiation. The gene-specific activator promotes the initiation of transcription through its interaction with one or more components of the transcriptional initiation machinery, hence leading to stimulated transcriptional initiation or activation. However, all activator proteins do not target the same component(s) of the transcriptional initiation machinery. Rather, they can have different target specificities, and thus, can lead to distinct mechanisms of transcriptional activation. Two such distinct mechanisms of transcriptional activation in yeast are mediated by the SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (Transcription factor IID) complexes, and are termed as "SAGA-dependent" and "TFIID-dependent" transcriptional activation, respectively. SAGA is the target of the activator in case of SAGA-dependent transcriptional activation, while the targeting of TFIID by the activator leads to TFIID-dependent transcriptional activation. Both the SAGA and TFIID complexes are highly conserved from yeast to human, and play crucial roles in gene activation among eukaryotes. The regulatory mechanisms of eukaryotic transcriptional activation by SAGA and TFIID are discussed here. This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!

  9. Rethinking transcriptional activation in the Arabidopsis circadian clock.

    Science.gov (United States)

    Fogelmark, Karl; Troein, Carl

    2014-07-01

    Circadian clocks are biological timekeepers that allow living cells to time their activity in anticipation of predictable daily changes in light and other environmental factors. The complexity of the circadian clock in higher plants makes it difficult to understand the role of individual genes or molecular interactions, and mathematical modelling has been useful in guiding clock research in model organisms such as Arabidopsis thaliana. We present a model of the circadian clock in Arabidopsis, based on a large corpus of published time course data. It appears from experimental evidence in the literature that most interactions in the clock are repressive. Hence, we remove all transcriptional activation found in previous models of this system, and instead extend the system by including two new components, the morning-expressed activator RVE8 and the nightly repressor/activator NOX. Our modelling results demonstrate that the clock does not need a large number of activators in order to reproduce the observed gene expression patterns. For example, the sequential expression of the PRR genes does not require the genes to be connected as a series of activators. In the presented model, transcriptional activation is exclusively the task of RVE8. Predictions of how strongly RVE8 affects its targets are found to agree with earlier interpretations of the experimental data, but generally we find that the many negative feedbacks in the system should discourage intuitive interpretations of mutant phenotypes. The dynamics of the clock are difficult to predict without mathematical modelling, and the clock is better viewed as a tangled web than as a series of loops.

  10. Development of transcriptional fusions to assess Leptospira interrogans promoter activity.

    Directory of Open Access Journals (Sweden)

    Gustavo M Cerqueira

    Full Text Available BACKGROUND: Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. METHODOLOGY AND PRINCIPAL FINDINGS: A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA and Sphingomyelinase 2 (sph2 promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. CONCLUSIONS: The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa.

  11. Analyzing phosphorylation-dependent regulation of subcellular localization and transcriptional activity of transcriptional coactivator NT-PGC-1α.

    Science.gov (United States)

    Chang, Ji Suk; Gettys, Thomas W

    2013-01-01

    Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a nuclear transcriptional coactivator that regulates the genes involved in energy metabolism. Recent evidence has been provided that alternative splicing of PPARGC1A gene produces a functional but predominantly cytosolic isoform of PGC-1α (NT-PGC-1α). We have demonstrated that transcriptional coactivation capacity of NT-PGC-1α is directly correlated with its nuclear localization in a PKA phosphorylation-dependent manner. In this chapter, we describe quantitative imaging analysis methods that are developed to measure the relative fluorescence intensity of the protein of interest in the nucleus and cytoplasm in a single cell and the frequency distribution of nuclear/cytoplasmic intensity ratios in the population of cells, respectively. This chapter also describes transient cotransfection and dual-luciferase reporter gene assay that examine the ability of coactivators to activate the transcriptional activity of transcription factors.

  12. Interaction of rice Bzip protein REB with the 5 '-upstream region of both rice sbel gene and waxy gene

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Rice starch branching enzyme 1 (SBE1) andgranule-bound starch synthase (GBSS) catalyzed the biosynthesis of amylopectin and amylose in rice developing endosperm respectively, and the genes encoding these two enmes, sbel and waxy, are mainly expressed in the endosperm. Within the 5 '-upstream region of rice sbel gene, we identified a 53 bp fragment C53 which could interact with the nuclear proteins extracted from rice endosperm. We also found that fragment Ha-2 in the 5'-upstream region of rice waxy gene could compete with C53 for its interaction. Further experiments demonstrated that rice bZIP protein REB could interact with two ACGT elements (G-box and Hex) in the fragment C53 as well as three ACGT elements (WG1,WG2, and WG3) in the fragment Ha-2. The WG1 element could compete well with Hex element for its interaction with REB. These results suggested that the expression of sbel and waxy gene in rice developing endosperm may be coordinately regulated by REB-like bZIP transcriptional factors.

  13. NCOA4 transcriptional coactivator inhibits activation of DNA replication origins.

    Science.gov (United States)

    Bellelli, Roberto; Castellone, Maria Domenica; Guida, Teresa; Limongello, Roberto; Dathan, Nina Alayne; Merolla, Francesco; Cirafici, Anna Maria; Affuso, Andrea; Masai, Hisao; Costanzo, Vincenzo; Grieco, Domenico; Fusco, Alfredo; Santoro, Massimo; Carlomagno, Francesca

    2014-07-01

    NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.

  14. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Ludmila R.P. Ferreira

    2008-03-01

    Full Text Available The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.A diferenciação de formas epimastigotas (proliferativas do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas, pode ser reproduzida em laborat

  15. The HMG-box mitochondrial transcription factor xl-mtTFA binds DNA as a tetramer to activate bidirectional transcription.

    OpenAIRE

    Antoshechkin, I; Bogenhagen, D F; Mastrangelo, I A

    1997-01-01

    The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided e...

  16. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    Energy Technology Data Exchange (ETDEWEB)

    Shlomai, Amir, E-mail: amirsh@tasmc.health.gov.il [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel); Institute for Gastroenterology and Liver disease, Tel-Aviv Sourasky Medical Center, 6 Weizmann street, Tel-Aviv (Israel); Shaul, Yosef [Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  17. Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons

    DEFF Research Database (Denmark)

    Mourier, Tobias; Willerslev, Eske

    2010-01-01

    transcriptional activity from Long Terminal Repeat (LTR) retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity...... of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes. CONCLUSIONS: Presumably, the host organism negatively regulates...... proliferation of LTR retrotransposons. The finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a posttranscriptional level. Alternatively, the transcriptional activity may signify a hitherto unrecognized activity level...

  18. WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes

    Directory of Open Access Journals (Sweden)

    Bol John F

    2011-05-01

    Full Text Available Abstract Background Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR. The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA. An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3 plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3. Results Expression studies with ICS1 promoter::β-glucuronidase (GUS genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA. Conclusions The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress

  19. Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity.

    Directory of Open Access Journals (Sweden)

    Juan Zhang

    Full Text Available Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBPα, peroxisome proliferators-activated receptor γ2 (PPARγ2, and other adipogenic genes in the process of adipogenesis. Berberine decreased cAMP-response element-binding protein (CREB phosphorylation and C/EBPβ expression at the early stage of 3T3-L1 preadipocyte differentiation. In addition, CREB phosphorylation and C/EBPβ expression induced by 3-isobutyl-1-methylxanthine (IBMX and forskolin were also attenuated by berberine. The binding activities of cAMP responsive element (CRE stimulated by IBMX and forskolin were inhibited by berberine. The binding of phosphorylated CREB to the promoter of C/EBPβ was abrogated by berberine after the induction of preadipocyte differentiation. These results suggest that berberine blocks adipogenesis mainly via suppressing CREB activity, which leads to a decrease in C/EBPβ-triggered transcriptional cascades.

  20. Regulation of transcription and activity of Rhizobium etli glutaminase A.

    Science.gov (United States)

    Huerta-Saquero, Alejandro; Calderón-Flores, Arturo; Díaz-Villaseñor, Andrea; Du Pont, Gisela; Durán, Socorro

    2004-08-01

    The present study determines the regulatory mechanisms that operate on Rhizobium etli glutaminase A. glsA gene expression levels were evaluated under several metabolic conditions by fusions of the glsA gene promoter and the transcriptional reporter cassette uidA2-aad. glsA expression was directly correlated to the glutaminase A activity found under the tested growth conditions, reaching its maximum level in the presence of glutamine and during exponential growth phase. Glutamine induces glsA expression. The influence of allosteric metabolites on glutaminase A activity was also determined. The purified enzyme was inhibited by 2-oxoglutarate and pyruvate, whereas oxaloacetate and glyoxylate modulate it positively. Glutaminase A is not inhibited by glutamate and is activated by ammonium. Glutaminase A participates in an ATP-consuming cycle where glutamine is continually degraded and resynthesized by glutamine synthetase (GS). GS and glutaminase A activities appear simultaneously during bacterial growth under different metabolic conditions and their control mechanisms are not reciprocal. Slight overproduction in glutaminase A expression causes a reduction in growth yield and a dramatic decrease in bacterial growth. We propose a model for regulation of glutaminase A, and discuss its contribution to glutamine cycle regulation. PMID:15279892

  1. Sucrose-induced translational repression of plant bZIP-type transcription factors

    NARCIS (Netherlands)

    Wiese, A.; Elzinga, N.; Wobbes, B.; Smeekens, S.

    2005-01-01

    Sugars as signalling molecules exert control on the transcription of many plant genes. Sugar signals also alter mRNA and protein stability. Increased sucrose concentrations specifically repress translation of the S-class basic region leucine zipper (bZIP) type transcription factor AtbZIP11/ATB2. Thi

  2. Cloned yeast and mammalian transcription factor TFIID gene products support basal but not activated metallothionein gene transcription

    International Nuclear Information System (INIS)

    Transcription factor IID (TFIID), the TATA binding factor, is thought to play a key role in the regulation of eukaryotic transcriptional initiation. The authors studied the role of TFIID in the transcription of the yeast metallothionein gene, which is regulated by the copper-dependent activator protein ACE1. Both basal and induced transcription of the metallothionein gene require TFIID and a functional TATA binding site. Crude human and mouse TFIID fractions, prepared from mammalian cells, respond to stimulation by ACE1, In contrast, human and yeast TFIID proteins expressed from the cloned genes do not respond to ACE1, except in the presence of what germ or yeast total cell extracts. These results indicate that the cloned TFIID gene products lack a component(s) or modifications(s) that is required for regulated as compared to basal transription

  3. DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pedersen, Jakob Madsen; Fredsøe, Jacob Christian; Rødgaard, Morten Terpager;

    2012-01-01

    To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-re...... transcriptional activation of genes with a repressible/inducible mode of regulation....

  4. The Cellular Bromodomain Protein Brd4 has Multiple Functions in E2-Mediated Papillomavirus Transcription Activation

    OpenAIRE

    Helfer, Christine M.; Junpeng Yan; Jianxin You

    2014-01-01

    The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription act...

  5. SUMO modification regulates the transcriptional activity of FLASH

    Energy Technology Data Exchange (ETDEWEB)

    Alm-Kristiansen, Anne Hege; Norman, Ingrid Louise; Matre, Vilborg [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway); Gabrielsen, Odd Stokke, E-mail: o.s.gabrielsen@imbv.uio.no [Department of Molecular Biosciences, University of Oslo, N-0316 Oslo (Norway)

    2009-09-25

    FLASH is a huge multifunctional nuclear protein that has been linked to apoptotic signalling, transcriptional control and Cajal body function. To gain further insight into the functions of the FLASH protein, we performed a yeast two-hybrid screening with FLASH as bait and identified the SUMO-conjugating enzyme Ubc9 as an interaction partner. The main interaction surface for Ubc9 was found in the C-terminal part of FLASH, which is also a target for sumoylation. We identified K1813 as the major sumoylation site in FLASH, being enhanced by the SUMO E3 ligases Pc2 and PIASy. Disruption of this SUMO-conjugation site did not change the speckled subnuclear localization of FLASH, but it caused a reduction in FLASH activity as measured in a Gal4-tethering assay. Interestingly, the SUMO-specific protease SENP1 activated FLASH in the same assay. Overall, our results point to a complex involvement of sumoylation in modulating the function of FLASH.

  6. Role of hippocampal activity-induced transcription in memory consolidation.

    Science.gov (United States)

    Eagle, Andrew L; Gajewski, Paula A; Robison, Alfred J

    2016-08-01

    Experience-dependent changes in the strength of connections between neurons in the hippocampus (HPC) are critical for normal learning and memory consolidation, and disruption of this process drives a variety of neurological and psychiatric diseases. Proper HPC function relies upon discrete changes in gene expression driven by transcription factors (TFs) induced by neuronal activity. Here, we describe the induction and function of many of the most well-studied HPC TFs, including cyclic-AMP response element binding protein, serum-response factor, AP-1, and others, and describe their role in the learning process. We also discuss the known target genes of many of these TFs and the purported mechanisms by which they regulate long-term changes in HPC synaptic strength. Moreover, we propose that future research in this field will depend upon unbiased identification of additional gene targets for these activity-dependent TFs and subsequent meta-analyses that identify common genes or pathways regulated by multiple TFs in the HPC during learning or disease. PMID:27180338

  7. Human transcriptional coactivator PC4 stimulates DNA end joining and activates DSB repair activity.

    Science.gov (United States)

    Batta, Kiran; Yokokawa, Masatoshi; Takeyasu, Kunio; Kundu, Tapas K

    2009-01-23

    Human transcriptional coactivator PC4 is a highly abundant nuclear protein that is involved in diverse cellular processes ranging from transcription to chromatin organization. Earlier, we have shown that PC4, a positive activator of p53, overexpresses upon genotoxic insult in a p53-dependent manner. In the present study, we show that PC4 stimulates ligase-mediated DNA end joining irrespective of the source of DNA ligase. Pull-down assays reveal that PC4 helps in the association of DNA ends through its C-terminal domain. In vitro nonhomologous end-joining assays with cell-free extracts show that PC4 enhances the joining of noncomplementary DNA ends. Interestingly, we found that PC4 activates double-strand break (DSB) repair activity through stimulation of DSB rejoining in vivo. Together, these findings demonstrate PC4 as an activator of nonhomologous end joining and DSB repair activity.

  8. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity.

    Science.gov (United States)

    Ahmed, Afsar U; Williams, Bryan R G; Hannigan, Gregory E

    2015-11-11

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding.

  9. Building gene expression signatures indicative of transcription factor activation to predict AOP modulation

    Science.gov (United States)

    Building gene expression signatures indicative of transcription factor activation to predict AOP modulation Adverse outcome pathways (AOPs) are a framework for predicting quantitative relationships between molecular initiatin...

  10. Activator control of nucleosome occupancy in activation and repression of transcription.

    Directory of Open Access Journals (Sweden)

    Gene O Bryant

    2008-12-01

    Full Text Available The relationship between chromatin structure and gene expression is a subject of intense study. The universal transcriptional activator Gal4 removes promoter nucleosomes as it triggers transcription, but how it does so has remained obscure. The reverse process, repression of transcription, has often been correlated with the presence of nucleosomes. But it is not known whether nucleosomes are required for that effect. A new quantitative assay describes, for any given location, the fraction of DNA molecules in the population that bears a nucleosome at any given instant. This allows us to follow the time courses of nucleosome removal and reformation, in wild-type and mutant cells, upon activation (by galactose and repression (by glucose of the GAL genes of yeast. We show that upon being freed of its inhibitor Gal80 by the action of galactose, Gal4 quickly recruits SWI/SNF to the genes, and that nucleosome "remodeler" rapidly removes promoter nucleosomes. In the absence of SWI/SNF, Gal4's action also results in nucleosome removal and the activation of transcription, but both processes are significantly delayed. Addition of glucose to cells growing in galactose represses transcription. But if galactose remains present, Gal4 continues to work, recruiting SWI/SNF and maintaining the promoter nucleosome-free despite it being repressed. This requirement for galactose is obviated in a mutant in which Gal4 works constitutively. These results show how an activator's recruiting function can control chromatin structure both during gene activation and repression. Thus, both under activating and repressing conditions, the activator can recruit an enzymatic machine that removes promoter nucleosomes. Our results show that whereas promoter nucleosome removal invariably accompanies activation, reformation of nucleosomes is not required for repression. The finding that there are two routes to nucleosome removal and activation of transcription-one that requires the

  11. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

    Science.gov (United States)

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2014-09-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2. PMID:24726898

  12. Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Lai Meng-Jiun

    2010-08-01

    Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

  13. Transcription factor PIF4 controls the thermosensory activation of flowering

    KAUST Repository

    Kumar, S. Vinod

    2012-03-21

    Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change. © 2012 Macmillan Publishers Limited. All rights reserved.

  14. Transcription factors expressed in soybean roots under drought stress.

    Science.gov (United States)

    Pereira, S S; Guimarães, F C M; Carvalho, J F C; Stolf-Moreira, R; Oliveira, M C N; Rolla, A A P; Farias, J R B; Neumaier, N; Nepomuceno, A L

    2011-10-21

    To gain insight into stress-responsive gene regulation in soybean plants, we identified consensus sequences that could categorize the transcription factors MYBJ7, BZIP50, C2H2, and NAC2 as members of the gene families myb, bzip, c2h2, and nac, respectively. We also investigated the evolutionary relationship of these transcription factors and analyzed their expression levels under drought stress. The NCBI software was used to find the predicted amino acid sequences of the transcription factors, and the Clustal X software was used to align soybean and other plant species sequences. Phylogenetic trees were built using the Mega 4.1 software by neighbor joining and the degree of confidence test by Bootstrap. Expression level studies were carried out using hydroponic culture; the experiments were designed in completely randomized blocks with three repetitions. The blocks consisted of two genotypes, MG/BR46 Conquista (drought-tolerant) and BR16 (drought-sensitive) and the treatments consisted of increasingly long dehydration periods (0, 25, 50, 75, and 100 min). The transcription factors presented domains and/or conserved regions that characterized them as belonging to the bzip, c2h2, myb, and nac families. Based on the phylogenetic trees, it was found that the myb, bzip and nac genes are closely related to myb78, bzip48 and nac2 of soybean and that c2h2 is closely related to c2h2 of Brassica napus. Expression of all genes was in general increased under drought stress in both genotypes. Major differences between genotypes were due to the lowering of the expression of the mybj7 and c2h2 genes in the drought-tolerant variety at some times. Over-expression or silencing of some of these genes has the potential to increase stress tolerance.

  15. Spi-1/PU.1 activates transcription through clustered DNA occupancy in erythroleukemia.

    Science.gov (United States)

    Ridinger-Saison, Maya; Boeva, Valentina; Rimmelé, Pauline; Kulakovskiy, Ivan; Gallais, Isabelle; Levavasseur, Benjamin; Paccard, Caroline; Legoix-Né, Patricia; Morlé, François; Nicolas, Alain; Hupé, Philippe; Barillot, Emmanuel; Moreau-Gachelin, Françoise; Guillouf, Christel

    2012-10-01

    Acute leukemias are characterized by deregulation of transcriptional networks that control the lineage specificity of gene expression. The aberrant overexpression of the Spi-1/PU.1 transcription factor leads to erythroleukemia. To determine how Spi-1 mechanistically influences the transcriptional program, we combined a ChIP-seq analysis with transcriptional profiling in cells from an erythroleukemic mouse model. We show that Spi-1 displays a selective DNA-binding that does not often cause transcriptional modulation. We report that Spi-1 controls transcriptional activation and repression partially through distinct Spi-1 recruitment to chromatin. We revealed several parameters impacting on Spi-1-mediated transcriptional activation. Gene activation is facilitated by Spi-1 occupancy close to transcriptional starting site of genes devoid of CGIs. Moreover, in those regions Spi-1 acts by binding to multiple motifs tightly clustered and with similar orientation. Finally, in contrast to the myeloid and lymphoid B cells in which Spi-1 exerts a physiological activity, in the erythroleukemic cells, lineage-specific cooperating factors do not play a prevalent role in Spi-1-mediated transcriptional activation. Thus, our work describes a new mechanism of gene activation through clustered site occupancy of Spi-1 particularly relevant in regard to the strong expression of Spi-1 in the erythroleukemic cells.

  16. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    Science.gov (United States)

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits. PMID:26134708

  17. Human ZCCHC12 activates AP-1 and CREB signaling as a transcriptional co-activator

    Institute of Scientific and Technical Information of China (English)

    Hong Li; Qian Liu; Xiang Hu; Du Feng; Shuanglin Xiang; Zhicheng He; Xingwang Hu; Jianlin Zhou; Xiaofeng Ding; Chang Zhou; Jian Zhang

    2009-01-01

    Mouse zinc finger CCHC domain containing 12 gene (ZCCHC12) has been identified as a transcriptional co-activator of bone morphogenetic protein (BMP) sig-naling,and human ZCCHC12 was reported to be related to non-syndromic X-linked mental retardation (NS-XLMR).However,the details of how human ZCCHCI2 involve in the NS-XLMR still remain unclear.In this study,we identified a novel nuclear localization signal (NLS) in the middle of human ZCCHC12 protein which is responsible for the nuclear localization.Multiple-tissue northern blot analysis indi-cated that ZCCHC12 is highly expressed in human brain.Furthermore,in situ hybridization showed that ZCCHC12 is specifically expressed in neuroepithelium of forebrain,midbrain,and diencephalon regions of mouse E10.5 embryos.Luciferase reporter assays demonstrated that ZCCHC12 enhanced the transcrip-tional activities of activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as a co-activator.In conclusion,we identified a new NLS in ZCCHC12 and figured out that ZCCHC12 functions as a transcriptional co-activator of AP-1 and CREB.

  18. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    Science.gov (United States)

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits.

  19. Transcriptional regulatory network triggered by oxidative signals configures the early response mechanisms of japonica rice to chilling stress

    KAUST Repository

    Yun, Kil-Young

    2010-01-25

    Background: The transcriptional regulatory network involved in low temperature response leading to acclimation has been established in Arabidopsis. In japonica rice, which can only withstand transient exposure to milder cold stress (10C), an oxidative-mediated network has been proposed to play a key role in configuring early responses and short-term defenses. The components, hierarchical organization and physiological consequences of this network were further dissected by a systems-level approach.Results: Regulatory clusters responding directly to oxidative signals were prominent during the initial 6 to 12 hours at 10C. Early events mirrored a typical oxidative response based on striking similarities of the transcriptome to disease, elicitor and wounding induced processes. Targets of oxidative-mediated mechanisms are likely regulated by several classes of bZIP factors acting on as1/ocs/TGA-like element enriched clusters, ERF factors acting on GCC-box/JAre-like element enriched clusters and R2R3-MYB factors acting on MYB2-like element enriched clusters.Temporal induction of several H2O2-induced bZIP, ERF and MYB genes coincided with the transient H2O2spikes within the initial 6 to 12 hours. Oxidative-independent responses involve DREB/CBF, RAP2 and RAV1 factors acting on DRE/CRT/rav1-like enriched clusters and bZIP factors acting on ABRE-like enriched clusters. Oxidative-mediated clusters were activated earlier than ABA-mediated clusters.Conclusion: Genome-wide, physiological and whole-plant level analyses established a holistic view of chilling stress response mechanism of japonica rice. Early response regulatory network triggered by oxidative signals is critical for prolonged survival under sub-optimal temperature. Integration of stress and developmental responses leads to modulated growth and vigor maintenance contributing to a delay of plastic injuries. 2010 Yun et al; licensee BioMed Central Ltd.

  20. Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein

    Science.gov (United States)

    Stankovic, B.; Vian, A.; Henry-Vian, C.; Davies, E.

    2000-01-01

    Localized wounding of one leaf in intact tomato (Lycopersicon esculentum Mill.) plants triggers rapid systemic transcriptional responses that might be involved in defense. To better understand the mechanism(s) of intercellular signal transmission in wounded tomatoes, and to identify the array of genes systemically up-regulated by wounding, a subtractive cDNA library for wounded tomato leaves was constructed. A novel cDNA clone (designated LebZIP1) encoding a DNA-binding protein was isolated and identified. This clone appears to be encoded by a single gene, and belongs to the family of basic leucine zipper domain (bZIP) transcription factors shown to be up-regulated by cold and dark treatments. Analysis of the mRNA levels suggests that the transcript for LebZIP1 is both organ-specific and up-regulated by wounding. In wounded wild-type tomatoes, the LebZIP1 mRNA levels in distant tissue were maximally up-regulated within only 5 min following localized wounding. Exogenous abscisic acid (ABA) prevented the rapid wound-induced increase in LebZIP1 mRNA levels, while the basal levels of LebZIP1 transcripts were higher in the ABA mutants notabilis (not), sitiens (sit), and flacca (flc), and wound-induced increases were greater in the ABA-deficient mutants. Together, these results suggest that ABA acts to curtail the wound-induced synthesis of LebZIP1 mRNA.

  1. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  2. A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

    Science.gov (United States)

    Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput...

  3. Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF

    NARCIS (Netherlands)

    Schuijers, Jurian; Mokry, Michal; Hatzis, Pantelis; Cuppen, Edwin; Clevers, Hans

    2014-01-01

    Active canonical Wnt signaling results in recruitment of β-catenin to DNA by TCF/LEF family members, leading to transcriptional activation of TCF target genes. However, additional transcription factors have been suggested to recruit β-catenin and tether it to DNA. Here, we describe the genome-wide p

  4. Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2014-03-01

    Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

  5. Genome-wide Expansion and Expression Divergence of the Basic Leucine Zipper Transcription Factors in Higher Plants with an Emphasis on Sorghum

    Institute of Scientific and Technical Information of China (English)

    Jizhou Wang; Junxia Zhou; Baolan Zhang; Jeevanandam Vanitha; Srinivasan Ramachandran; Shu-Ye Jiang

    2011-01-01

    Plant bZIP transcription factors play crucial roles in multiple biological processes. However,little is known about the sorghum bZIP gene family although the sorghum genome has been completely sequenced. In this study,we have carried out a genome-wide identification and characterization of this gene family in sorghum.Our data show that the genome encodes at least 92 bZIP transcription factors. These bZIP genes have been expanded mainly by segmental duplication. Such an expansion mechanism has also been observed in rice,arabidopsis and many other plant organisms,suggesting a common expansion mode of this gene family in plants. Further investigation shows that most of the bZIP members have been present in the most recent common ancestor of sorghum and rice and the major expansion would occur before the sorghum-rice split era. Although these bZIP genes have been duplicated with a long history,they exhibited limited functional divergence as shown by nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) analyses. Their retention was mainly due to the high percentages of expression divergence. Our data also showed that this gene family might play a role in multiple developmental stages and tissues and might be regarded as important regulators of various abiotic stresses and sugar signaling.

  6. Transcriptional coactivator CIITA, a functional homolog of TAF1, has kinase activity.

    Science.gov (United States)

    Soe, Katherine C; Devaiah, Ballachanda N; Singer, Dinah S

    2013-11-01

    The Major Histocompatibility Complex (MHC) class II transactivator (CIITA) mediates activated immune responses and its deficiency results in the Type II Bare Lymphocyte Syndrome. CIITA is a transcriptional co-activator that regulates γ-interferon-activated transcription of MHC class I and class II genes. It is also a functional homolog of TAF1, a component of the general transcription factor complex TFIID. TAF1 and CIITA both possess intrinsic acetyltransferase (AT) activity that is required for transcription initiation. In response to induction by γ-interferon, CIITA and it's AT activity bypass the requirement for TAF1 AT activity. TAF1 also has kinase activity that is essential for its function. However, no similar activity has been identified for CIITA thus far. Here we report that CIITA, like TAF1, is a serine-threonine kinase. Its substrate specificity parallels, but does not duplicate, that of TAF1 in phosphorylating the TFIID component TAF7, the RAP74 subunit of the general transcription factor TFIIF and histone H2B. Like TAF1, CIITA autophosphorylates, affecting its interaction with TAF7. Additionally, CIITA phosphorylates histone H2B at Ser36, a target of TAF1 that is required for transcription during cell cycle progression and stress response. However, unlike TAF1, CIITA also phosphorylates all the other histones. The identification of this novel kinase activity of CIITA further clarifies its role as a functional homolog of TAF1 which may operate during stress and γ-IFN activated MHC gene transcription.

  7. Prediction of Pathway Activation by Xenobiotic-Responsive Transcription Factors in the Mouse Liver

    Science.gov (United States)

    Many drugs and environmentally-relevant chemicals activate xenobioticresponsive transcription factors (TF). Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening. Starting with a large compendium of Affymet...

  8. EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors

    Directory of Open Access Journals (Sweden)

    Florkowska Magdalena

    2012-03-01

    Full Text Available Abstract Background Tristetraprolin (TTP is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described. Results Our results demonstrate the induction of the gene coding TTP (ZFP36 by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp. Conclusions EGF activates TTP expression via ELK-1 and EGR-1 transcription factors.

  9. MED16 and MED23 of Mediator are coactivators of lipopolysaccharide- and heat-shock-induced transcriptional activators

    OpenAIRE

    Kim, Tae Whan; Kwon, Yong-Jae; Kim, Jung Mo; Song, Young-Hwa; Kim, Se Nyun; Kim, Young-Joon

    2004-01-01

    Transcriptional activators interact with diverse proteins and recruit transcriptional machinery to the activated promoter. Recruitment of the Mediator complex by transcriptional activators is usually the key step in transcriptional activation. However, it is unclear how Mediator recognizes different types of activator proteins. To systematically identify the subunits responsible for the signal- and activator-specific functions of Mediator in Drosophila melanogaster, each Mediator subunit was ...

  10. Analysis of p53 mutants for transcriptional activity.

    OpenAIRE

    Raycroft, L.; Schmidt, J. R.; Yoas, K; Hao, M M; Lozano, G.

    1991-01-01

    The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 protein...

  11. O-GlcNAc modification of Sp3 and Sp4 transcription factors negatively regulates their transcriptional activities.

    Science.gov (United States)

    Ha, Changhoon; Lim, Kihong

    2015-11-13

    The addition of O-linked N-acetylglucosamine (O-GlcNAc) on serine or threonine modifies a myriad of proteins and regulates their function, stability and localization. O-GlcNAc modification is common among chromosome-associated proteins, such as transcription factors, suggesting its extensive involvement in gene expression regulation. In this study, we demonstrate the O-GlcNAc status of the Sp family members of transcription factors and the functional impact on their transcriptional activities. We highlight the presence of O-GlcNAc residues in Sp3 and Sp4, but not Sp2, as demonstrated by their enrichment in GlcNAc positive protein fractions and by detection of O-GlcNAc residues on Sp3 and Sp4 co-expressed in Escherichia coli together with O-GlcNAc transferase (OGT) using an O-GlcNAc-specific antibody. Deletion mutants of Sp3 and Sp4 indicate that the majority of O-GlcNAc sites reside in their N-terminal transactivation domain. Overall, using reporter gene assays and co-immunoprecipitations, we demonstrate a functional inhibitory role of O-GlcNAc modifications in Sp3 and Sp4 transcription factors. Thereby, our study strengthens the current notion that O-GlcNAc modification is an important regulator of protein interactome.

  12. PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation.

    OpenAIRE

    Kannan, P; Yu, Y; Wankhade, S; Tainsky, M A

    1999-01-01

    Overexpression of transcription factor AP-2 has been implicated in the tumorigenicity of the human teratocarcinoma cell lines PA-1 that contain an activated ras oncogene. Here we show evidence that overexpression of AP-2 sequesters transcriptional coactivators which results in self-inhibition. We identified AP-2-interacting proteins and determined whether these proteins were coactivators for AP-2-mediated transcription. One such interacting protein is polyADP-ribose polymerase (PARP). PARP su...

  13. Exercise-Induced VEGF Transcriptional Activation in Brain, Lung and Skeletal Muscle

    OpenAIRE

    Tang, Kechun; Xia, Feng Cheng; Wagner, Peter D.; Breen, Ellen C.

    2009-01-01

    Muscle VEGF expression is upregulated by exercise. Whether this VEGF response is regulated by transcription and/or post-transcriptional mechanisms is unknown. Hypoxia may be responsible: myocyte PO2 falls greatly during exercise and VEGF is a hypoxia-responsive gene. Whether exercise induces VEGF expression in other organs important to acute physical activity is also unknown. To address these questions, we created a VEGF/Luciferase reporter mouse and measured VEGF transcription, mRNA and prot...

  14. Synergistic transcriptional activation by one regulatory protein in response to two metabolites

    OpenAIRE

    Bundy, Becky M.; Collier, Lauren S.; Hoover, Timothy R.; Neidle, Ellen L.

    2002-01-01

    BenM is a LysR-type bacterial transcriptional regulator that controls aromatic compound degradation in Acinetobacter sp. ADP1. Here, in vitro transcription assays demonstrated that two metabolites of aromatic compound catabolism, benzoate and cis,cis-muconate, act synergistically to activate gene expression. The level of BenM-regulated benA transcription was significantly higher in response to both compounds than the combined levels due to each alone. These compounds also were more effective ...

  15. Multiple MAPK cascades regulate the transcription of IME1, the master transcriptional activator of meiosis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kahana-Edwin, Smadar; Stark, Michal; Kassir, Yona

    2013-01-01

    The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UAS)ru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i) The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA) pathway, targeting the transcription factors (TFs), Com2 and Sko1; (ii) high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK) pathway and its corresponding TF Sko1; (iii) elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv) the nitrogen source repressed UASru activity through Sum1; and (v) the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis. PMID:24236068

  16. Multiple MAPK cascades regulate the transcription of IME1, the master transcriptional activator of meiosis in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Smadar Kahana-Edwin

    Full Text Available The choice between alternative developmental pathways is primarily controlled at the level of transcription. Induction of meiosis in budding yeasts in response to nutrient levels provides a system to investigate the molecular basis of cellular decision-making. In Saccharomyces cerevisiae, entry into meiosis depends on multiple signals converging upon IME1, the master transcriptional activator of meiosis. Here we studied the regulation of the cis-acting regulatory element Upstream Activation Signal (UASru, which resides within the IME1 promoter. Guided by our previous data acquired using a powerful high-throughput screening system, here we provide evidence that UASru is regulated by multiple stimuli that trigger distinct signal transduction pathways as follows: (i The glucose signal inhibited UASru activity through the cyclic AMP (cAMP/protein kinase A (PKA pathway, targeting the transcription factors (TFs, Com2 and Sko1; (ii high osmolarity activated UASru through the Hog1/mitogen-activated protein kinase (MAPK pathway and its corresponding TF Sko1; (iii elevated temperature increased the activity of UASru through the cell wall integrity pathway and the TFs Swi4/Mpk1 and Swi4/Mlp1; (iv the nitrogen source repressed UASru activity through Sum1; and (v the absence of a nitrogen source was detected and transmitted to UASru by the Kss1 and Fus3 MAPK pathways through their respective downstream TFs, Ste12/Tec1 and Ste12/Ste12 as well as by their regulators Dig1/2. These signaling events were specific to UASru; they did not affect the mating and filamentation response elements that are regulated by MAPK pathways. The complex regulation of UASru through all the known vegetative MAPK pathways is unique to S. cerevisiae and is specific for IME1, likely because it is the master regulator of gametogenesis.

  17. The cellular bromodomain protein Brd4 has multiple functions in E2-mediated papillomavirus transcription activation.

    Science.gov (United States)

    Helfer, Christine M; Yan, Junpeng; You, Jianxin

    2014-08-01

    The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription activation, is important for E2's transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2's interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+), a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV) life cycle. PMID:25140737

  18. The Cellular Bromodomain Protein Brd4 has Multiple Functions in E2-Mediated Papillomavirus Transcription Activation

    Directory of Open Access Journals (Sweden)

    Christine M. Helfer

    2014-08-01

    Full Text Available The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb, a functional interaction partner of Brd4 in transcription activation, is important for E2’s transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2’s interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+, a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV life cycle.

  19. Clinical application of transcriptional activators of bile salt transporters ☆

    OpenAIRE

    Baghdasaryan, Anna; Chiba, Peter; Trauner, Michael

    2014-01-01

    Hepatobiliary bile salt (BS) transporters are critical determinants of BS homeostasis controlling intracellular concentrations of BSs and their enterohepatic circulation. Genetic or acquired dysfunction of specific transport systems causes intrahepatic and systemic retention of potentially cytotoxic BSs, which, in high concentrations, may disturb integrity of cell membranes and subcellular organelles resulting in cell death, inflammation and fibrosis. Transcriptional regulation of canalicular...

  20. Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons

    Directory of Open Access Journals (Sweden)

    Willerslev Eske

    2010-03-01

    Full Text Available Abstract Background Retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. Less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed - or near fixation - in the population or strain from which genetic material has been extracted for sequencing. One pre-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from Long Terminal Repeat (LTR retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity was analysed for both full-length LTR retrotransposons and solitary LTRs. Results Two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription

  1. Inferring yeast cell cycle regulators and interactions using transcription factor activities

    Directory of Open Access Journals (Sweden)

    Galbraith Simon J

    2005-06-01

    Full Text Available Abstract Background Since transcription factors are often regulated at the post-transcriptional level, their activities, rather than expression levels may provide valuable information for investigating functions and their interactions. The recently developed Network Component Analysis (NCA and its generalized form (gNCA provide a robust framework for deducing the transcription factor activities (TFAs from various types of DNA microarray data and transcription factor-gene connectivity. The goal of this work is to demonstrate the utility of TFAs in inferring transcription factor functions and interactions in Saccharomyces cerevisiae cell cycle regulation. Results Using gNCA, we determined 74 TFAs from both wild type and fkh1 fkh2 deletion mutant microarray data encompassing 1529 ORFs. We hypothesized that transcription factors participating in the cell cycle regulation exhibit cyclic activity profiles. This hypothesis was supported by the TFA profiles of known cell cycle factors and was used as a basis to uncover other potential cell cycle factors. By combining the results from both cluster analysis and periodicity analysis, we recovered nearly 90% of the known cell cycle regulators, and identified 5 putative cell cycle-related transcription factors (Dal81, Hap2, Hir2, Mss11, and Rlm1. In addition, by analyzing expression data from transcription factor knockout strains, we determined 3 verified (Ace2, Ndd1, and Swi5 and 4 putative interaction partners (Cha4, Hap2, Fhl1, and Rts2 of the forkhead transcription factors. Sensitivity of TFAs to connectivity errors was determined to provide confidence level of these predictions. Conclusion By subjecting TFA profiles to analyses based upon physiological signatures we were able to identify cell cycle related transcription factors consistent with current literature, transcription factors with potential cell cycle dependent roles, and interactions between transcription factors.

  2. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    Science.gov (United States)

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  3. Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts.

    Science.gov (United States)

    Pandian, Ganesh N; Taniguchi, Junichi; Junetha, Syed; Sato, Shinsuke; Han, Le; Saha, Abhijit; AnandhaKumar, Chandran; Bando, Toshikazu; Nagase, Hiroki; Vaijayanthi, Thangavel; Taylor, Rhys D; Sugiyama, Hiroshi

    2014-01-24

    The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation.

  4. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors.

    Science.gov (United States)

    Morbitzer, Robert; Römer, Patrick; Boch, Jens; Lahaye, Thomas

    2010-12-14

    Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNA-binding specificity of transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of user-defined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated the desired promoters in a sequence-specific manner as mutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NK mediates specific interaction with G nucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes.

  5. Signal transducer and activator of transcription 6 gene G2964A polymorphism and inflammatory bowel disease.

    NARCIS (Netherlands)

    Xia, B; Crusius, J.B.A.; Wu, J; Zwiers, A.; Bodegraven, van A.A.; Pena, A.S.

    2003-01-01

    Signal transducer and activator of transcription 6 (STAT6) is a key transcription factor involved in interleukin 4 (IL-4) and IL-13-mediated Th2 response. The STAT6 gene is located on chromosome 12q13.3-14.1 (IBD2 region) and is therefore a positional and functional candidate gene for study in infla

  6. Enhanced transcriptional activation by E2 proteins from the oncogenic human papillomaviruses.

    OpenAIRE

    Kovelman, R; Bilter, G K; Glezer, E; Tsou, A Y; Barbosa, M S

    1996-01-01

    A systematic comparison of transcriptional activation by papillomavirus E2 proteins revealed that the E2 proteins from high-risk human papillomaviruses (human papillomavirus type 16 [HPV-16] and HPV-18) are much more active than are the E2 proteins from low-risk HPVs (HPV-6b and HPV-11). Despite the tropism of HPVs for particular epithelial cell types, this difference in transcriptional activation was observed in a number of different epithelial and nonepithelial cells. The enhanced activitie...

  7. Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

    OpenAIRE

    Groscurth Peter; Dumrese Claudia; Sundstrom Hanna; Walch Michael; Latinovic-Golic Sonja; Ziegler Urs

    2007-01-01

    Abstract Background Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522) were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. Results In this study we investigated generatio...

  8. DNA-recognition by a σ54 transcriptional activator from Aquifex aeolicus

    OpenAIRE

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G.; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E.

    2014-01-01

    Transcription initiation by bacterial σ54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) f...

  9. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    International Nuclear Information System (INIS)

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73

  10. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Kasim, Vivi, E-mail: vivikasim78@gmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Yang, Li [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Miyagishi, Makoto [Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566 (Japan); Wu, Shourong, E-mail: shourongwu@hotmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  11. TBP Domain Symmetry in Basal and Activated Archaeal Transcription

    OpenAIRE

    Ouhammouch, Mohamed; Hausner, Winfried; Geiduschek, E Peter

    2008-01-01

    The TATA-box binding protein (TBP) is the platform for assembly of archaeal and eukaryotic transcription preinitiation complexes. Ancestral gene duplication and fusion events have produced the saddle-shaped TBP molecule, with its two direct-repeat subdomains and pseudo-two-fold symmetry. Collectively, eukaryotic TBPs have diverged from their present-day archaeal counterparts, which remain highly symmetrical. The similarity of the N- and C-halves of archaeal TBPs is especially pronounced in th...

  12. Variable Glutamine-Rich Repeats Modulate Transcription Factor Activity

    OpenAIRE

    Gemayel, Rita; Chavali, Sreenivas; Pougach, Ksenia; Legendre, Matthieu; Zhu, Bo; Boeynaems, Steven; van der Zande, Elisa; Gevaert, Kris; Rousseau, Frederic; Schymkowitz, Joost; Babu, M Madan; Verstrepen, Kevin J.

    2015-01-01

    Summary Excessive expansions of glutamine (Q)-rich repeats in various human proteins are known to result in severe neurodegenerative disorders such as Huntington’s disease and several ataxias. However, the physiological role of these repeats and the consequences of more moderate repeat variation remain unknown. Here, we demonstrate that Q-rich domains are highly enriched in eukaryotic transcription factors where they act as functional modulators. Incremental changes in the number of repeats i...

  13. Thioredoxin interacting protein inhibits hypoxia-inducible factor transcriptional activity

    OpenAIRE

    Farrell, Michael R; Rogers, Lynette K.; Liu, Yusen; Welty, Stephen E.; Tipple, Trent E.

    2010-01-01

    Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. ...

  14. SENSITIVE TO PROTON RHIZOTOXICITY1, CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2, and other transcription factors are involved in ALUMINUM-ACTIVATED MALATE TRANSPORTER1 expression.

    Science.gov (United States)

    Tokizawa, Mutsutomo; Kobayashi, Yuriko; Saito, Tatsunori; Kobayashi, Masatomo; Iuchi, Satoshi; Nomoto, Mika; Tada, Yasuomi; Yamamoto, Yoshiharu Y; Koyama, Hiroyuki

    2015-03-01

    In Arabidopsis (Arabidopsis thaliana) the root apex is protected from aluminum (Al) rhizotoxicity by excretion of malate, an Al chelator, by ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1). AtALMT1 expression is fundamentally regulated by the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) zinc finger protein, but other transcription factors have roles that enable Al-inducible expression with a broad dynamic range. In this study, we characterized multiple cis-elements in the AtALMT1 promoter that interact with transcription factors. In planta complementation assays of AtALMT1 driven by 5' truncated promoters of different lengths showed that the promoter region between -540 and 0 (the first ATG) restored the Al-sensitive phenotype of atalm1 and thus contains cis-elements essential for AtALMT1 expression for Al tolerance. Computation of overrepresented octamers showed that eight regions in this promoter region contained potential cis-elements involved in Al induction and STOP1 regulation. Mutation in a position around -297 from the first ATG completely inactivated AtALMT1 expression and Al response. In vitro binding assays showed that this region contained the STOP1 binding site, which accounted for the recognition by four zinc finger domains of the protein. Other positions were characterized as cis-elements that regulated expression by repressors and activators and a transcription factor that determines root tip expression of AtALMT1. From the consensus of known cis-elements, we identified CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR2 to be an activator of AtALMT1 expression. Al-inducible expression of AtALMT1 changed transcription starting sites, which increased the abundance of transcripts with a shortened 5' untranslated region. The present analyses identified multiple mechanisms that regulate AtALMT1 expression.

  15. Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression.

    Science.gov (United States)

    Building predictive gene signatures through simultaneous assessment of transcription factor activation and gene expression Exposure to many drugs and environmentally-relevant chemicals can cause adverse outcomes. These adverse outcomes, such as cancer, have been linked to mol...

  16. Two distinct domains of Flo8 activator mediates its role in transcriptional activation and the physical interaction with Mss11.

    Science.gov (United States)

    Kim, Hye Young; Lee, Sung Bae; Kang, Hyen Sam; Oh, Goo Taeg; Kim, TaeSoo

    2014-06-27

    Flo8 is a transcriptional activator essential for the inducible expression of a set of target genes such as STA1, FLO11, and FLO1 encoding an extracellular glucoamylase and two cell surface proteins, respectively. However, the molecular mechanism of Flo8-mediated transcriptional activation remains largely elusive. By generating serial deletion constructs, we revealed here that a novel transcriptional activation domain on its extreme C-terminal region plays a crucial role in activating transcription. On the other hand, the N-terminal LisH motif of Flo8 appears to be required for its physical interaction with another transcriptional activator, Mss11, for their cooperative transcriptional regulation of the shared targets. Additionally, GST pull-down experiments uncovered that Flo8 and Mss11 can directly form either a heterodimer or a homodimer capable of binding to DNA, and we also showed that this formed complex of two activators interacts functionally and physically with the Swi/Snf complex. Collectively, our findings provide valuable clues for understanding the molecular mechanism of Flo8-mediated transcriptional control of multiple targets.

  17. (-)-Epicatechin gallate (ECG) stimulates osteoblast differentiation via Runt-related transcription factor 2 (RUNX2) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated transcriptional activation.

    Science.gov (United States)

    Byun, Mi Ran; Sung, Mi Kyung; Kim, A Rum; Lee, Cham Han; Jang, Eun Jung; Jeong, Mi Gyeong; Noh, Minsoo; Hwang, Eun Sook; Hong, Jeong-Ho

    2014-04-01

    Osteoporosis is a degenerative bone disease characterized by low bone mass and is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. It is known that the bioactive compounds present in green tea increase osteogenic activity and decrease the risk of fracture by improving bone mineral density. However, the detailed mechanism underlying these beneficial effects has yet to be elucidated. In this study, we investigated the osteogenic effect of (-)-epicatechin gallate (ECG), a major bioactive compound found in green tea. We found that ECG effectively stimulates osteoblast differentiation, indicated by the increased expression of osteoblastic marker genes. Up-regulation of osteoblast marker genes is mediated by increased expression and interaction of the transcriptional coactivator with PDZ-binding motif (TAZ) and Runt-related transcription factor 2 (RUNX2). ECG facilitates nuclear localization of TAZ through PP1A. PP1A is essential for osteoblast differentiation because inhibition of PP1A activity was shown to suppress ECG-mediated osteogenic differentiation. Taken together, the results showed that ECG stimulates osteoblast differentiation through the activation of TAZ and RUNX2, revealing a novel mechanism for green tea-stimulated osteoblast differentiation.

  18. Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha

    DEFF Research Database (Denmark)

    Hwang, C S; Mandrup, S; MacDougald, O A;

    1996-01-01

    Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob......, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter....

  19. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    OpenAIRE

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  20. Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor.

    OpenAIRE

    Cavaillès, V; Dauvois, S; L'Horset, F; Lopez, G; Hoare, S.; Kushner, P J; Parker, M G

    1995-01-01

    A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti-estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen re...

  1. Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene.

    Science.gov (United States)

    Kim, Kihoon; Kim, AeRi

    2010-09-01

    Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR.

  2. Four Arabidopsis AREB/ABF transcription factors function predominantly in gene expression downstream of SnRK2 kinases in abscisic acid signalling in response to osmotic stress.

    Science.gov (United States)

    Yoshida, Takuya; Fujita, Yasunari; Maruyama, Kyonoshin; Mogami, Junro; Todaka, Daisuke; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2015-01-01

    Under osmotic stress conditions such as drought and high salinity, the plant hormone abscisic acid (ABA) plays important roles in stress-responsive gene expression mainly through three bZIP transcription factors, AREB1/ABF2, AREB2/ABF4 and ABF3, which are activated by SNF1-related kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). However, since the three AREB/ABFs are crucial, but not exclusive, for the SnRK2-mediated gene expression, transcriptional pathways governed by SRK2D/E/I are not fully understood. Here, we show that a bZIP transcription factor, ABF1, is a functional homolog of AREB1, AREB2 and ABF3 in ABA-dependent gene expression in Arabidopsis. Despite lower expression levels of ABF1 than those of the three AREB/ABFs, the areb1 areb2 abf3 abf1 mutant plants displayed increased sensitivity to drought and decreased sensitivity to ABA in primary root growth compared with the areb1 areb2 abf3 mutant. Genome-wide transcriptome analyses revealed that expression of downstream genes of SRK2D/E/I, which include many genes functioning in osmotic stress responses and tolerance such as transcription factors and LEA proteins, was mostly impaired in the quadruple mutant. Thus, these results indicate that the four AREB/ABFs are the predominant transcription factors downstream of SRK2D/E/I in ABA signalling in response to osmotic stress during vegetative growth.

  3. Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells.

    Science.gov (United States)

    Sozio, Margaret S; Lu, Changyue; Zeng, Yan; Liangpunsakul, Suthat; Crabb, David W

    2011-10-01

    AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) are critical regulators of short-term and long-term fatty acid oxidation, respectively. We examined whether the activities of these molecules were coordinately regulated. H4IIEC3 cells were transfected with PPAR-α and PPAR-γ expression plasmids and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid. The cells were treated with PPAR agonists (WY-14,643 and rosiglitazone), AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin, and the AMPK inhibitor compound C. Both AICAR and metformin decreased basal and WY-14,643-stimulated PPAR-α activity; compound C increased agonist-stimulated reporter activity and partially reversed the effect of the AMPK activators. Similar effects on PPAR-γ were seen, with both AICAR and metformin inhibiting PPRE reporter activity. Compound C increased basal PPAR-γ activity and rosiglitazone-stimulated activity. In contrast, retinoic acid receptor-α (RAR-α), another nuclear receptor that dimerizes with retinoid X receptor (RXR), was largely unaffected by the AMPK activators. Compound C modestly increased AM580 (an RAR agonist)-stimulated activity. The AMPK activators did not affect PPAR-α binding to DNA, and there was no consistent correlation between effects of the AMPK activators and inhibitor on PPAR and the nuclear localization of AMPK-α subunits. Expression of either a constitutively active or dominant negative AMPK-α inhibited basal and WY-14,643-stimulated PPAR-α activity and basal and rosiglitazone-stimulated PPAR-γ activity. We concluded that the AMPK activators AICAR and metformin inhibited transcriptional activities of PPAR-α and PPAR-γ, whereas inhibition of AMPK with compound C activated both PPARs. The effects of AMPK do not appear to be mediated through effects on RXR or on PPAR/RXR binding to DNA. These effects are independent of kinase activity and instead appear to

  4. Highly asynchronous and asymmetric cleavage divisions accompany early transcriptional activity in pre-blastula medaka embryos.

    Directory of Open Access Journals (Sweden)

    Michael Kraeussling

    Full Text Available In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT. Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.

  5. Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

    Science.gov (United States)

    House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

    2015-06-01

    Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

  6. DNA Topoisomerases maintain promoters in a state competent for transcriptional activation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jakob Madsen Pedersen

    Full Text Available To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence, and enrichment of a nucleosome at a critical position in the promoter region, in line with a repressible/inducible mode of regulation. Single-gene studies with a range of genes belonging to this group demonstrate that topoisomerases play an important role during activation of these genes. Subsequent in-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation and elongation of PHO5, as well as the nuclear entrance of Pho4p. Finally, we provide evidence that topoisomerases are required to maintain the PHO5 promoter in a superhelical state, which is competent for proper activation. In conclusion, our results reveal a hitherto unknown function of topoisomerases during transcriptional activation of genes with a repressible/inducible mode of regulation.

  7. Encoding four gene expression programs in the activation dynamics of a single transcription factor.

    Science.gov (United States)

    Hansen, Anders S; O'Shea, Erin K

    2016-04-01

    Cellular signaling response pathways often exhibit a bow-tie topology [1,2]: multiple upstream stress signals converge on a single shared transcription factor, which is thought to induce different downstream gene expression programs (Figure 1A). However, if several different signals activate the same transcription factor, can each signal then induce a specific gene expression response? A growing body of literature supports a temporal coding theory where information about environmental signals can be encoded, at least partially, in the temporal dynamics of the shared transcription factor [1,2]. For example, in the case of the budding yeast transcription factor Msn2, different stresses induce distinct Msn2 activation dynamics: Msn2 shows pulsatile nuclear activation with dose-dependent frequency under glucose limitation, but sustained nuclear activation with dose-dependent amplitude under oxidative stress [3]. These dynamic patterns can then lead to differential gene expression responses [3-5], but it is not known how much specificity can be obtained. Thus, a major question of this temporal coding theory is how many gene response programs or cellular functions can be robustly encoded by dynamic control of a single transcription factor. Here we provide the first direct evidence that, simply by regulating the activation dynamics of a single transcription factor, it is possible to preferentially induce four distinct gene expression programs. PMID:27046808

  8. Enhanced osteoclastogenesis by mitochondrial retrograde signaling through transcriptional activation of the cathepsin K gene.

    Science.gov (United States)

    Guha, Manti; Srinivasan, Satish; Koenigstein, Alexander; Zaidi, Mone; Avadhani, Narayan G

    2016-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its end point. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca(2) ](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages, this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Ctsk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Ctsk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Ctsk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss.

  9. Withaferin A inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells

    OpenAIRE

    Lee, Joomin; Hahm, Eun-Ryeong; Singh, Shivendra V

    2010-01-01

    We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of human breast cancer cells in culture and in vivo in association with apoptosis induction. The present study builds on these observations and demonstrates that WA inhibits constitutive as well as interleukin-6 (IL-6)-inducible activation of signal transducer and activator of transcription 3 (STAT3), which is an oncogenic transcription factor act...

  10. Interaction of the transcription start site core region and transcription factor YY1 determine ascorbate transporter SVCT2 exon 1a promoter activity.

    Directory of Open Access Journals (Sweden)

    Huan Qiao

    Full Text Available Transcription of the ascorbate transporter, SVCT2, is driven by two distinct promoters in exon 1 of the transporter sequence. The exon 1a promoter lacks a classical transcription start site and little is known about regulation of promoter activity in the transcription start site core (TSSC region. Here we present evidence that the TSSC binds the multifunctional initiator-binding protein YY1. Electrophoresis shift assays using YY1 antibody showed that YY1 is present as one of two major complexes that specifically bind to the TSSC. The other complex contains the transcription factor NF-Y. Mutations in the TSSC that decreased YY1 binding also impaired the exon 1a promoter activity despite the presence of an upstream activating NF-Y/USF complex, suggesting that YY1 is involved in the regulation of the exon 1a transcription. Furthermore, YY1 interaction with NF-Y and/or USF synergistically enhanced the exon 1a promoter activity in transient transfections and co-activator p300 enhanced their synergistic activation. We propose that the TSSC plays a vital role in the exon 1a transcription and that this function is partially carried out by the transcription factor YY1. Moreover, co-activator p300 might be able to synergistically enhance the TSSC function via a "bridge" mechanism with upstream sequences.

  11. In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.

    Science.gov (United States)

    Lin, Shuailiang; Ewen-Campen, Ben; Ni, Xiaochun; Housden, Benjamin E; Perrimon, Norbert

    2015-10-01

    A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.

  12. Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2)

    NARCIS (Netherlands)

    Takano, E.; Gramajo, H.C.; Strauch, E.; White, J.; Bibb, M.J.

    1992-01-01

    Transcription of redD, the activator gene required for production of the red-pigmented antibiotic undecylprodigiosin by Streptomyces coelicolor A3(2), showed a dramatic increase during the transition from exponential to stationary phase. The increase in redD expression was followed by transcription

  13. Nucleosome distortion as a possible mechanism of transcription activation domain function.

    Science.gov (United States)

    Erkina, Tamara Y; Erkine, Alexandre M

    2016-01-01

    After more than three decades since the discovery of transcription activation domains (ADs) in gene-specific activators, the mechanism of their function remains enigmatic. The widely accepted model of direct recruitment by ADs of co-activators and basal transcriptional machinery components, however, is not always compatible with the short size yet very high degree of sequence randomness and intrinsic structural disorder of natural and synthetic ADs. In this review, we formulate the basis for an alternative and complementary model, whereby sequence randomness and intrinsic structural disorder of ADs are necessary for transient distorting interactions with promoter nucleosomes, triggering promoter nucleosome translocation and subsequently gene activation. PMID:27679670

  14. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

    Directory of Open Access Journals (Sweden)

    Julia K Bialek

    Full Text Available CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs, act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR, for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

  15. Phosphatidylserine enhances IKBKAP transcription by activating the MAPK/ERK signaling pathway.

    Science.gov (United States)

    Donyo, Maya; Hollander, Dror; Abramovitch, Ziv; Naftelberg, Shiran; Ast, Gil

    2016-04-01

    Familial dysautonomia (FD) is a genetic disorder manifested due to abnormal development and progressive degeneration of the sensory and autonomic nervous system. FD is caused by a point mutation in the IKBKAP gene encoding the IKAP protein, resulting in decreased protein levels. A promising potential treatment for FD is phosphatidylserine (PS); however, the manner by which PS elevates IKAP levels has yet to be identified. Analysis of ChIP-seq results of the IKBKAP promoter region revealed binding of the transcription factors CREB and ELK1, which are regulated by the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) signaling pathway. We show that PS treatment enhanced ERK phosphorylation in cells derived from FD patients. ERK activation resulted in elevated IKBKAP transcription and IKAP protein levels, whereas pretreatment with the MAPK inhibitor U0126 blocked elevation of the IKAP protein level. Overexpression of either ELK1 or CREB activated the IKBKAP promoter, whereas downregulation of these transcription factors resulted in a decrease of the IKAP protein. Additionally, we show that PS improves cell migration, known to be enhanced by MAPK/ERK activation and abrogated in FD cells. In conclusion, our results demonstrate that PS activates the MAPK/ERK signaling pathway, resulting in activation of transcription factors that bind the promoter region of IKBKAP and thus enhancing its transcription. Therefore, compounds that activate the MAPK/ERK signaling pathway could constitute potential treatments for FD. PMID:26769675

  16. Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells

    DEFF Research Database (Denmark)

    Friedrichsen, Birgitte N; Richter, Henrijette E; Hansen, Johnny A;

    2003-01-01

    cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation......-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate...

  17. Conserved interaction of the papillomavirus E2 transcriptional activator proteins with human and yeast TFIIB proteins.

    OpenAIRE

    Benson, J D; Lawande, R; Howley, P M

    1997-01-01

    Papillomavirus early gene expression is regulated by the virus gene-encoded E2 proteins. The best-characterized E2 protein, encoded by bovine papillomavirus type 1 (BPV-1), has been shown to interact with basal transcription factor IIB (TFIIB) and the TATA binding protein basal transcription factor (N. M. Rank and P. F. Lambert, J. Virol. 69:6323-6334, 1995). We demonstrate that the potent E2 transcriptional activator protein encoded by a gene of human PV type 16 also interacts with TFIIB in ...

  18. Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts.

    OpenAIRE

    Keyse, S M; Applegate, L. A.; Tromvoukis, Y; Tyrrell, R M

    1990-01-01

    Treatment of cultured human skin fibroblasts with near-UV radiation, hydrogen peroxide, and sodium arsenite induces accumulation of heme oxygenase mRNA and protein. In this study, these treatments led to a dramatic increase in the rate of RNA transcription from the heme oxygenase gene but had no effect on mRNA stability. Transcriptional activation, therefore, appears to be the major mechanism of stimulation of expression of this gene by either oxidative stress or sulfydryl reagents.

  19. The myogenic regulatory gene Mef2 is a direct target for transcriptional activation by Twist during Drosophila myogenesis

    OpenAIRE

    Cripps, Richard M.; Black, Brian L.; Zhao, Bin; Lien, Ching-Ling; Schulz, Robert A.; Olson, Eric N.

    1998-01-01

    MEF2 is a MADS-box transcription factor required for muscle development in Drosophila. Here, we show that the bHLH transcription factor Twist directly regulates Mef2 expression in adult somatic muscle precursor cells via a 175-bp enhancer located 2245 bp upstream of the transcriptional start site. Within this element, a single evolutionarily conserved E box is essential for enhancer activity. Twist protein can bind to this E box to activate Mef2 transcription, and ectopic expression of twist ...

  20. Full p53 transcriptional activation potential is dispensable for tumor suppression in diverse lineages.

    Science.gov (United States)

    Jiang, Dadi; Brady, Colleen A; Johnson, Thomas M; Lee, Eunice Y; Park, Eunice J; Scott, Matthew P; Attardi, Laura D

    2011-10-11

    Over half of all human cancers, of a wide variety of types, sustain mutations in the p53 tumor suppressor gene. Although p53 limits tumorigenesis through the induction of apoptosis or cell cycle arrest, its molecular mechanism of action in tumor suppression has been elusive. The best-characterized p53 activity in vitro is as a transcriptional activator, but the identification of numerous additional p53 biochemical activities in vitro has made it unclear which mechanism accounts for tumor suppression. Here, we assess the importance of transcriptional activation for p53 tumor suppression function in vivo in several tissues, using a knock-in mouse strain expressing a p53 mutant compromised for transcriptional activation, p53(25,26). p53(25,26) is severely impaired for the transactivation of numerous classical p53 target genes, including p21, Noxa, and Puma, but it retains the ability to activate a small subset of p53 target genes, including Bax. Surprisingly, p53(25,26) can nonetheless suppress tumor growth in cancers derived from the epithelial, mesenchymal, central nervous system, and lymphoid lineages. Therefore, full transactivation of most p53 target genes is dispensable for p53 tumor suppressor function in a range of tissue types. In contrast, a transcriptional activation mutant that is completely defective for transactivation, p53(25,26,53,54), fails to suppress tumor development. These findings demonstrate that transcriptional activation is indeed broadly critical for p53 tumor suppressor function, although this requirement reflects the limited transcriptional activity observed with p53(25,26) rather than robust transactivation of a full complement of p53 target genes.

  1. Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics

    KAUST Repository

    Roy, S.

    2015-06-27

    Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

  2. Transcriptional Activity of rRNA Genes in Barley Cells after Mutagenic Treatment.

    Science.gov (United States)

    Kwasniewska, Jolanta; Jaskowiak, Joanna

    2016-01-01

    In the present study, the combination of the micronucleus test with analysis of the activity of the rRNA genes in mutagen-treated Hordeum vulgare (barley) by maleic hydrazide (MH) cells was performed. Simultaneously fluorescence in situ hybridization (FISH) with 25S rDNA as probes and an analysis of the transcriptional activity of 35S rRNA genes with silver staining were performed. The results showed that transcriptional activity is always maintained in the micronuclei although they are eliminated during the next cell cycle. The analysis of the transcriptional activity was extended to barley nuclei. MH influenced the fusion of the nucleoli in barley nuclei. The silver staining enabled detection of the nuclear bodies which arose after MH treatment. The results confirmed the usefulness of cytogenetic techniques in the characterization of micronuclei. Similar analyses can be now extended to other abiotic stresses to study the response of plant cells to the environment. PMID:27257817

  3. Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

    Science.gov (United States)

    Thakore, Pratiksha I; Gersbach, Charles A

    2016-01-01

    Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

  4. Weak estrogenic transcriptional activities of Bisphenol A and Bisphenol S

    OpenAIRE

    GRIGNARD ELISE; Bremer, Susanne; LAPENNA SILVIA

    2011-01-01

    In 2011, the European Commission has restricted the use of Bisphenol A in plastic infant feeding bottles. In a response to this restriction, Bisphenol S is now often used as a component of plastic substitutes for the production of babybottles. One of the major concerns leading to the restriction of Bisphenol A was its weak estrogenic activity. By using two highly standardised transactivation assays, we could demonstrate that the estrogenic activity of Bisphenol A and Bisphenol S i...

  5. Activity-dependent transport of the transcriptional coactivator CRTC1 from synapse to nucleus.

    Science.gov (United States)

    Ch'ng, Toh Hean; Uzgil, Besim; Lin, Peter; Avliyakulov, Nuraly K; O'Dell, Thomas J; Martin, Kelsey C

    2012-07-01

    Long-lasting changes in synaptic efficacy, such as those underlying long-term memory, require transcription. Activity-dependent transport of synaptically localized transcriptional regulators provides a direct means of coupling synaptic stimulation with changes in transcription. The CREB-regulated transcriptional coactivator (CRTC1), which is required for long-term hippocampal plasticity, binds CREB to potently promote transcription. We show that CRTC1 localizes to synapses in silenced hippocampal neurons but translocates to the nucleus in response to localized synaptic stimulation. Regulated nuclear translocation occurs only in excitatory neurons and requires calcium influx and calcineurin activation. CRTC1 is controlled in a dual fashion with activity regulating CRTC1 nuclear translocation and cAMP modulating its persistence in the nucleus. Neuronal activity triggers a complex change in CRTC1 phosphorylation, suggesting that CRTC1 may link specific types of stimuli to specific changes in gene expression. Together, our results indicate that synapse-to-nuclear transport of CRTC1 dynamically informs the nucleus about synaptic activity.

  6. A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

    Directory of Open Access Journals (Sweden)

    Tsai Chueh-Jen

    2010-01-01

    Full Text Available Abstract There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α and nuclear factor-kappa B (NF-κB were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.

  7. The dynamic changes of DNA methylation and histone modifications of salt responsive transcription factor genes in soybean.

    Directory of Open Access Journals (Sweden)

    Yuguang Song

    Full Text Available Epigenetic modification contributes to the regulation of gene expression and plant development under salinity stress. Here we describe the identification of 49 soybean transcription factors by microarray analysis as being inducible by salinity stress. A semi-quantitative RT-PCR-based expression assay confirmed the salinity stress inducibility of 45 of these 49 transcription factors, and showed that ten of them were up-regulated when seedlings were exposed to the demethylation agent 5-aza-2-deoxycytidine. Salinity stress was shown to affect the methylation status of four of these ten transcription factors (one MYB, one b-ZIP and two AP2/DREB family members using a combination of bisulfite sequencing and DNA methylation-sensitive DNA gel blot analysis. ChIP analysis indicated that the activation of three of the four DNA methylated transcription factors was correlated with an increased level of histone H3K4 trimethylation and H3K9 acetylation, and/or a reduced level of H3K9 demethylation in various parts of the promoter or coding regions. Our results suggest a critical role for some transcription factors' activation/repression by DNA methylation and/or histone modifications in soybean tolerance to salinity stress.

  8. Poised transcription factories prime silent uPA gene prior to activation.

    Directory of Open Access Journals (Sweden)

    Carmelo Ferrai

    2010-01-01

    Full Text Available The position of genes in the interphase nucleus and their association with functional landmarks correlate with active and/or silent states of expression. Gene activation can induce chromatin looping from chromosome territories (CTs and is thought to require de novo association with transcription factories. We identify two types of factory: "poised transcription factories," containing RNA polymerase II phosphorylated on Ser5, but not Ser2, residues, which differ from "active factories" associated with phosphorylation on both residues. Using the urokinase-type plasminogen activator (uPA gene as a model system, we find that this inducible gene is predominantly associated with poised (S5p(+S2p(- factories prior to activation and localized at the CT interior. Shortly after induction, the uPA locus is found associated with active (S5p(+S2p(+ factories and loops out from its CT. However, the levels of gene association with poised or active transcription factories, before and after activation, are independent of locus positioning relative to its CT. RNA-FISH analyses show that, after activation, the uPA gene is transcribed with the same frequency at each CT position. Unexpectedly, prior to activation, the uPA loci internal to the CT are seldom transcriptionally active, while the smaller number of uPA loci found outside their CT are transcribed as frequently as after induction. The association of inducible genes with poised transcription factories prior to activation is likely to contribute to the rapid and robust induction of gene expression in response to external stimuli, whereas gene positioning at the CT interior may be important to reinforce silencing mechanisms prior to induction.

  9. Weak estrogenic transcriptional activities of Bisphenol A and Bisphenol S.

    Science.gov (United States)

    Grignard, Elise; Lapenna, Silvia; Bremer, Susanne

    2012-08-01

    In 2011, the European Commission has restricted the use of Bisphenol A in plastic infant feeding bottles. In a response to this restriction, Bisphenol S is now often used as a component of plastic substitutes for the production of babybottles. One of the major concerns leading to the restriction of Bisphenol A was its weak estrogenic activity. By using two highly standardised transactivation assays, we could demonstrate that the estrogenic activity of Bisphenol A and Bisphenol S is of a comparable potency. Furthermore, some insights about the structure-activity relationships of these two chemicals and their metabolites could be gained from in silico predictions of their relative estrogen receptor-binding affinities and their liver phase-I biotransformation.

  10. Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

    OpenAIRE

    Hori, R; Pyo, S.; Carey, M

    1995-01-01

    Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB...

  11. Transcriptional factors, Mafs and their biological roles

    Institute of Scientific and Technical Information of China (English)

    Mariko Tsuchiya; Ryoichi Misaka; Kosaku Nitta; Ken Tsuchiya

    2015-01-01

    The Maf family of transcription factors is characterizedby a typical bZip structure; these transcription factorsact as important regulators of the development anddifferentiation of many organs and tissues, includingthe kidney. The Maf family consists of two subgroupsthat are characterized according to their structure largeMaf transcription factors and small Maf transcriptionfactors. The large Maf subgroup consists of fourproteins, designated as MAFA, MAFB, c-MAF and neuralretina-specific leucine zipper. In particular, MAFA is adistinct molecule that has been attracting the attentionof researchers because it acts as a strong transactivatorof insulin, suggesting that Maf transcription factors arelikely to be involved in systemic energy homeostasis. Inthis review, we focused on the regulation of glucose/energy balance by Maf transcription factors in variousorgans.

  12. Differences in transcriptional activity of cutaneous human papillomaviruses

    DEFF Research Database (Denmark)

    Vasiljevic, Natasa; Nielsen, Lone; Doherty, Geoff;

    2008-01-01

    The interaction between UV-B irradiation and cutaneous human papillomaviruses (HPV) has been suggested to be of relevance for the development of non-melanoma skin cancers. We investigated the activity within the upstream regulatory region (URR) of the HPV types 8, 38, 92, 93 and 96, as well as th...

  13. The JmjC domain of Gis1 is dispensable for transcriptional activation.

    Science.gov (United States)

    Yu, Yao; Neiman, Aaron M; Sternglanz, Rolf

    2010-11-01

    Yeast Gis1 protein functions as a transcription factor after nutrient limitation and oxidative stress. In this report, we show that Gis1 also regulates the induction of several genes involved in spore wall synthesis during sporulation. Gis1 contains a JmjC domain near its N-terminus. In many proteins, JmjC domains provide histone demethylase activity. Whether the JmjC domain of Gis1 contributes to its transcriptional activation is still unknown. Here, we show that gis1 point mutations that abolish Fe (II) and α-ketoglutarate binding, known cofactors in other JmjC proteins, are still able to induce transcription normally during glucose starvation and sporulation. Even the deletion of the entire JmjC domain does not affect transcriptional activation by Gis1. Moreover, the JmjC domain is not required for the toxicity associated with Gis1 overexpression. The data demonstrate that the JmjC domain is dispensable for transcriptional activation by Gis1 during nutrient stress and sporulation.

  14. Elk3 from hamster--a ternary complex factor with strong transcriptional repressor activity.

    Science.gov (United States)

    Hjortoe, Gertrud Malene; Weilguny, Dietmar; Willumsen, Berthe Marie

    2005-01-01

    Elk3 belongs to the Ets family of transcription factors, which are regulated by the Ras/mitogen-activated protein kinase-signaling pathway. In the absence of Ras, this protein is a strong inhibitor of transcription and may be directly involved in regulation of growth by downregulating the transcription of genes that are activated during entry into G1. We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c-fos promoter, Elk3 from CHO cells seems to inhibit other promoters controlling expression of proteins involved in G1/S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicate that hamster Elk3 is a target of the Ras-Raf-MAPK pathway, and cotransfections with constitutively active H-ras relieves its negative transcriptional activity. No cells stably expressing exogenous Elk3 could be obtained, possibly due to an unspecified toxic or growth retarding effect. These findings support a possible role for Elk3 in growth regulation and reveal a high degree of homology for this protein across species. PMID:15684718

  15. A non canonical subtilase attenuates the transcriptional activation of defence responses in Arabidopsis thaliana

    Science.gov (United States)

    Serrano, Irene; Buscaill, Pierre; Audran, Corinne; Pouzet, Cécile; Jauneau, Alain; Rivas, Susana

    2016-01-01

    Proteases play crucial physiological functions in all organisms by controlling the lifetime of proteins. Here, we identified an atypical protease of the subtilase family [SBT5.2(b)] that attenuates the transcriptional activation of plant defence independently of its protease activity. The SBT5.2 gene produces two distinct transcripts encoding a canonical secreted subtilase [SBT5.2(a)] and an intracellular protein [SBT5.2(b)]. Concomitant to SBT5.2(a) downregulation, SBT5.2(b) expression is induced after bacterial inoculation. SBT5.2(b) localizes to endosomes where it interacts with and retains the defence-related transcription factor MYB30. Nuclear exclusion of MYB30 results in its reduced transcriptional activation and, thus, suppressed resistance. sbt5.2 mutants, with abolished SBT5.2(a) and SBT5.2(b) expression, display enhanced defence that is suppressed in a myb30 mutant background. Moreover, overexpression of SBT5.2(b), but not SBT5.2(a), in sbt5.2 plants reverts the phenotypes displayed by sbt5.2 mutants. Overall, we uncover a regulatory mode of the transcriptional activation of defence responses previously undescribed in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19755.001 PMID:27685353

  16. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.

    Directory of Open Access Journals (Sweden)

    Takashi Watanabe

    Full Text Available Mastermind-like 1 (MAML1 is a transcriptional co-activator in the Notch signaling pathway. Recently, however, several reports revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. We found that MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2, a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2 from the Runx2-regulated osteocalcin gene promoter. Interestingly, a deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Moreover, inhibition of Notch signaling did not affect the action of MAML1 on Runx2, suggesting that the activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1(-/- embryos at embryonic day 16.5 (E16.5 had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1 and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1 expression, a marker of hypertrophic chondrocyte, were observed. These observations suggest that chondrocyte maturation was impaired in MAML1(-/- mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.

  17. Non-Canonical EZH2 Transcriptionally Activates RelB in Triple Negative Breast Cancer

    Science.gov (United States)

    Lawrence, Cortney L.; Baldwin, Albert S.

    2016-01-01

    Enhancer of zeste homology 2 (EZH2) is the methyltransferase component of the polycomb repressive complex (PRC2) which represses gene transcription via histone H3 trimethylation at lysine 23 (H3K27me3). EZH2 activity has been linked with oncogenesis where it is thought to block expression of certain tumor suppressors. Relative to a role in cancer, EZH2 functions to promote self-renewal and has been shown to be important for the tumor-initiating cell (TIC) phenotype in breast cancer. Recently a non-canonical role for EZH2 has been identified where it promotes transcriptional activation of certain genes. Here we show that EZH2, through a methyltransferase-independent mechanism, promotes the transcriptional activation of the non-canonical NF-κB subunit RelB to drive self-renewal and the TIC phenotype of triple-negative breast cancer cells. PMID:27764181

  18. Nutritional conditions regulate transcriptional activity of SF-1 by controlling sumoylation and ubiquitination.

    Science.gov (United States)

    Lee, Jiwon; Yang, Dong Joo; Lee, Syann; Hammer, Gary D; Kim, Ki Woo; Elmquist, Joel K

    2016-01-11

    Steroidogenic factor 1 (SF-1) is a transcription factor expressed in the ventral medial nucleus of the hypothalamus that regulates energy homeostasis. However, the molecular mechanisms of SF-1 in the control of energy balance are largely unknown. Here, we show that nutritional conditions, such as the presence or absence of serum, affect SF-1 action. Serum starvation significantly decreased hypothalamic SF-1 levels by promoting ubiquitin-dependent degradation, and sumoylation was required for this process. SF-1 transcriptional activity was also differentially regulated by nutritional status. Under normal conditions, the transcriptional activity of hypothalamic SF-1 was activated by SUMO, but this was attenuated during starvation. Taken together, these results indicate that sumoylation and ubiquitination play crucial roles in the regulation of SF-1 function and that these effects are dependent on nutritional conditions, further supporting the importance of SF-1 in the control of energy homeostasis.

  19. Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo.

    Science.gov (United States)

    Liu, Junbo; Ma, Jun

    2015-11-01

    The Drosophila embryo at the mid-blastula transition (MBT) concurrently experiences a receding first wave of zygotic transcription and the surge of a massive second wave. It is not well understood how genes in the first wave become turned off transcriptionally and how their precise timing may impact embryonic development. Here we perturb the timing of the shutdown of Bicoid (Bcd)-dependent hunchback (hb) transcription in the embryo through the use of a Bcd mutant that has heightened activating potency. A delayed shutdown specifically increases Bcd-activated hb levels, and this alters spatial characteristics of the patterning outcome and causes developmental defects. Our study thus documents a specific participation of maternal activator input strength in the timing of molecular events in precise accordance with MBT morphological progression. PMID:26395487

  20. Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity

    OpenAIRE

    Juan Zhang; Hongju Tang; Ruyuan Deng; Ning Wang; Yuqing Zhang; Yao Wang; Yun Liu; Fengying Li; Xiao Wang; Libin Zhou

    2015-01-01

    Berberine, one of the major constituents of Chinese herb Rhizoma coptidis, has been demonstrated to lower blood glucose, blood lipid, and body weight in patients with type 2 diabetes mellitus. The anti-obesity effect of berberine has been attributed to its anti-adipogenic activity. However, the underlying molecular mechanism remains largely unknown. In the present study, we found that berberine significantly suppressed the expressions of CCAAT/enhancer-binding protein (C/EBP)α, peroxisome pro...

  1. NACK is an integral component of the Notch transcriptional activation complex and is critical for development and tumorigenesis.

    Science.gov (United States)

    Weaver, Kelly L; Alves-Guerra, Marie-Clotilde; Jin, Ke; Wang, Zhiqiang; Han, Xiaoqing; Ranganathan, Prathibha; Zhu, Xiaoxia; DaSilva, Thiago; Liu, Wei; Ratti, Francesca; Demarest, Renee M; Tzimas, Cristos; Rice, Meghan; Vasquez-Del Carpio, Rodrigo; Dahmane, Nadia; Robbins, David J; Capobianco, Anthony J

    2014-09-01

    The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, Notch activation complex kinase (NACK), which acts as a Notch transcriptional coactivator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are coexpressed during mouse development and that homozygous loss of NACK is embryonic lethal. Finally, we show that NACK is also a Notch target gene, establishing a feed-forward loop. Thus, our data indicate that NACK is a key component of the Notch transcriptional complex and is an essential regulator of Notch-mediated tumorigenesis and development.

  2. PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma

    LENUS (Irish Health Repository)

    Keld, R

    2011-06-28

    Background: Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells.\\r\

  3. Alterations in leukocyte transcriptional control pathway activity associated with major depressive disorder and antidepressant treatment.

    Science.gov (United States)

    Mellon, S H; Wolkowitz, O M; Schonemann, M D; Epel, E S; Rosser, R; Burke, H B; Mahan, L; Reus, V I; Stamatiou, D; Liew, C-C; Cole, S W

    2016-01-01

    Major depressive disorder (MDD) is associated with a significantly elevated risk of developing serious medical illnesses such as cardiovascular disease, immune impairments, infection, dementia and premature death. Previous work has demonstrated immune dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress (nuclear factor erythroid-derived 2-like 2, NFE2l2 or NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1-4 (EGR1-4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators. PMID:27219347

  4. Alterations in leukocyte transcriptional control pathway activity associated with major depressive disorder and antidepressant treatment

    Science.gov (United States)

    Mellon, S H; Wolkowitz, O M; Schonemann, M D; Epel, E S; Rosser, R; Burke, H B; Mahan, L; Reus, V I; Stamatiou, D; Liew, C -C; Cole, S W

    2016-01-01

    Major depressive disorder (MDD) is associated with a significantly elevated risk of developing serious medical illnesses such as cardiovascular disease, immune impairments, infection, dementia and premature death. Previous work has demonstrated immune dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress (nuclear factor erythroid-derived 2-like 2, NFE2l2 or NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1–4 (EGR1–4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators. PMID:27187237

  5. Nucleophosmin contributes to the transcriptional activation function of the Epstein-Barr virus EBNA1 protein.

    Science.gov (United States)

    Malik-Soni, Natasha; Frappier, Lori

    2014-02-01

    The Epstein-Barr virus (EBV) EBNA1 protein plays important roles in latent infection, including transcriptional activation of EBV latency genes by binding to the family-of-repeats (FR) element. Through a proteomic approach, we previously identified an interaction between EBNA1 and the histone chaperone nucleophosmin. Here we show that the EBNA1-nucleophosmin interaction is direct and requires the Gly-Arg-rich sequences that contribute to transactivation. Additionally, nucleophosmin is recruited by EBNA1 to the FR element and is required for EBNA1-mediated transcriptional activation.

  6. Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells.

    Science.gov (United States)

    Clark, Sara; Rainville, Jennifer; Zhao, Xing; Katzenellenbogen, Benita S; Pfaff, Donald; Vasudevan, Nandini

    2014-01-01

    While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17β-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Gαq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERα phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERα is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

  7. Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation.

    Science.gov (United States)

    Xin, Qi-Liang; Qiu, Jing-Tao; Cui, Sheng; Xia, Guo-Liang; Wang, Hai-Bin

    2016-08-25

    Estrogen receptor (ER) and progesterone receptor (PR) are two important members of steroid receptors family, an evolutionarily conserved family of transcription factors. Upon binding to their ligands, ER and PR enter cell nucleus to interact with specific DNA element in the context of chromatin to initiate the transcription of diverse target genes, which largely depends on the timely recruitment of a wide range of cofactors. Moreover, the interactions between steroid hormones and their respective receptors also trigger post-translational modifications on these receptors to fine-tune their transcriptional activities. Besides the well-known phosphorylation modifications on tyrosine and serine/threonine residues, recent studies have identified several other covalent modifications, such as ubiquitylation and sumoylation. These post-translational modifications of steroid receptors affect its stability, subcellular localization, and/or cofactor recruitment; eventually influence the duration and extent of transcriptional activation. This review is to focus on the recent research progress on the transcriptional activation of nuclear ER and PR as well as their physiological functions in early pregnancy, which may help us to better understand related female reproductive diseases. PMID:27546504

  8. SUMOylation can regulate the activity of ETS-like transcription factor 4.

    Science.gov (United States)

    Kaikkonen, Sanna; Makkonen, Harri; Rytinki, Miia; Palvimo, Jorma J

    2010-08-01

    ETS-like transcription factor 4 (ELK4) (a.k.a. serum response factor accessory protein 1) belongs to the ternary complex factor (TCF) subfamily of E twenty-six (ETS) domain transcription factors. Compared to the other TCF subfamily members, ELK1 and ELK3 (NET), there is limited information of the mechanisms regulating the ELK4 activity. Here, we show that the ELK4 can be covalently modified (SUMOylated) by small ubiquitin-related modifier (SUMO) 1 protein, an important regulator of signaling and transcription. SUMOylation of ELK4 was reversed by SUMO-specific proteases (SENP) 1 and 2 and stimulated by SUMO E3 ligase PIAS3. Conserved lysine residue 167 that is located in the NET inhibitory domain of ELK4 was identified as the main site of SUMO-1 conjugation. Interestingly, mutation of the K167 disrupting the SUMOylation markedly enhanced the transcriptional activity of the ELK4, but weakened its repressive function on c-fos promoter. In conclusion, our results suggest that covalent modification by SUMO-1 can regulate the activity of ELK4, contributing to the transcriptional repression by the ELK4. PMID:20637912

  9. Transcription of Mammalian cis-Regulatory Elements Is Restrained by Actively Enforced Early Termination.

    Science.gov (United States)

    Austenaa, Liv M I; Barozzi, Iros; Simonatto, Marta; Masella, Silvia; Della Chiara, Giulia; Ghisletti, Serena; Curina, Alessia; de Wit, Elzo; Bouwman, Britta A M; de Pretis, Stefano; Piccolo, Viviana; Termanini, Alberto; Prosperini, Elena; Pelizzola, Mattia; de Laat, Wouter; Natoli, Gioacchino

    2015-11-01

    Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs. PMID:26593720

  10. Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

    Science.gov (United States)

    Wood, Lauren W; Cox, Nicole I; Phelps, Cody A; Lai, Shao-Chiang; Poddar, Arjun; Talbot, Conover; Mu, David

    2016-01-01

    Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1(+) lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1(+) lung cancer cells (designated EDM-TTF-1(+)) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1(+) conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1(+) lung adenocarcinomas. PMID:26912193

  11. Effect of protein kinase C inhibitor (PKCI) on radiation sensitivity and c-fos transcription activity

    International Nuclear Information System (INIS)

    The human genetic disorder ataxia-telangiectasia (AT) is a multisystem disease characterized by extreme radiosensitivity. The recent identification of the gene mutated in AT, ATM, and the demonstration that it encodes a homologous domain of phosphatidylinositol 3-kinase (PI3-K), the catalytic subunit of an enzyme involved in transmitting signals from the cell surface to the nucleus, provide support for a role of this gene in signal transduction. Although ionizing radiation was known to induce c-fos transcription, nothing is known about how ATM or PKCI mediated signal transduction pathway modulates the c-fos gene transcription and gene expression. Here we have studied the effect of PKCI on radiation sensitivity and c-fos transcription in normal and AT cells. Normal (LM217) and AT (AT58IVA) cells were transfected with PKCI expression plasmid and the overexpression and integration of PKCI was evaluated by northern blotting and polymerase chain reaction, respectively. 5 Gy of radiation was exposed to LM and AT cells transfected with PKCI expression plasmid and cells were harvested 48 hours after radiation and investigated apoptosis with TUNEL method. The c-fos transcription activity was studied by performing CAT assay of reporter gene after transfection of c-fos CAT plasmid into AT and LM cells. Our results demonstrate for the first time a role of PKCI on. the radiation sensitivity and c-fos expression in LM and AT cells. PKCI increased radiation induced apoptosis in LM cells but reduced apoptosis in AT cells. The basal c-fos transcription activity is 70 times lower in AT cells than that in LM cells. The c-fos transcription activity was repressed by overexpression of PKCI in LM cells but not in AT cells. After induction of c-fos by Ras protein, overexpression of PKCI repressed c-fos transcription in LM cells but not in AT cells. Overexpression of PKCI increased radiation sensitivity and repressed c-fos transcription in LM cells but not in AT cells. The results may be a

  12. Model of transcriptional activation by MarA in escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Wall, Michael E [Los Alamos National Laboratory; Rosner, Judah L [NATIONAL INSTITUTE OF HEALTH; Martin, Robert G [NATIONAL INSTITUTE OF HEALTH

    2009-01-01

    The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

  13. Expression of Activated Epidermal Growth Factor Receptor and Transcription Factor E2F in Condyloma Accuminata

    Institute of Scientific and Technical Information of China (English)

    俞小虹; 程浩; 郑伟

    2003-01-01

    Objective: To study the expression of activated epi-dermal growth factor receptor (EGFR) and transcrip-tion factor E2F (E2F) in Condyloma Accuminata(CA) patients. Methods: Immunofluorescent techniques were used to investigate the expression of activated EGFR and E2F in CA patients. Results: The expression of activated EGFR on the membrane of epithelial cells in CA lesions was sig-nificantly greater compared to expression levers in the control group (P<0.01). Moreover, the co-expres-sion of activated EGFR and E2F was significantly in-creased compared to the control group (P<0.01).Conclusion: Our observations suggest that the in-crease in activated EGFR expression may stimulate hyperplasia in CA patients through the activation of transcription factor E2F.

  14. Lysine methylation of HIV-1 Tat regulates transcriptional activity of the viral LTR

    Directory of Open Access Journals (Sweden)

    Flynn Elizabeth K

    2008-05-01

    Full Text Available Abstract Background The rate of transcription of the HIV-1 viral genome is mediated by the interaction of the viral protein Tat with the LTR and other transcriptional machinery. These specific interactions can be affected by the state of post-translational modifications on Tat. Previously, we have shown that Tat can be phosphorylated and acetylated in vivo resulting in an increase in the rate of transcription. In the present study, we investigated whether Tat could be methylated on lysine residues, specifically on lysine 50 and 51, and whether this modification resulted in a decrease of viral transcription from the LTR. Results We analyzed the association of Tat with histone methyltransferases of the SUV39-family of SET domain containing proteins in vitro. Tat was found to associate with both SETDB1 and SETDB2, two enzymes which exhibit methyltransferase activity. siRNA against SETDB1 transfected into cell systems with both transient and integrated LTR reporter genes resulted in an increase in transcription of the HIV-LTR in the presence of suboptimal levels of Tat. In vitro methylation assays with Tat peptides containing point mutations at lysines 50 and 51 showed an increased incorporation of methyl groups on lysine 51, however, both residues indicated susceptibility for methylation. Conclusion The association of Tat with histone methyltransferases and the ability for Tat to be methylated suggests an interesting mechanism of transcriptional regulation through the recruitment of chromatin remodeling proteins to the HIV-1 promoter.

  15. PEA3activates CXCL12transcription in MCF-7breast cancer cells%PEA3 activates CXCL12 transcription in MCF-7 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Li; CHEN Bo-bin; LI Jun-jie; JIN Wei; SHAO Zhi-min

    2011-01-01

    Objective To explore the activity of PEA3 ( polyomavirus enhancer activator 3 ) on CXCL12 (Chemokine CXC motif ligand 12) transcription and to reveal the role of PEA3 involved in CXCL12-mediated metastasis and angiogenesis in breast cancer. Methods Methods such as cell transfection, ChIP assay (chromatin immunoprecipitation ), and siRNA (small interfering RNA) were applied to demonstrate and confirm the interaction between PEA3 and CXCL12. Results Over-expression of PEA3 could increase the CXCL12 mRNA level and the CXCL12 promoter activity in human MCF-7 breast cancer cells. ChIP assay demonstrated that PEA3 could bind to the CXCL12 promoter in the cells transfected with PEA3 expression vector. PEA3 siRNA decreased CXCL12 promoter activity and the binding of PEA3 to the CXCL12 promoter in MCF-7 cells. Conclusions PEA3 could activate CXCL12 promoter transcription. It may be a potential mechanism of tumor angiogenesis and metastasis regarding of PEA3 and CXCL12.

  16. TRIM24 Is an Oncogenic Transcriptional Activator in Prostate Cancer.

    Science.gov (United States)

    Groner, Anna C; Cato, Laura; de Tribolet-Hardy, Jonas; Bernasocchi, Tiziano; Janouskova, Hana; Melchers, Diana; Houtman, René; Cato, Andrew C B; Tschopp, Patrick; Gu, Lei; Corsinotti, Andrea; Zhong, Qing; Fankhauser, Christian; Fritz, Christine; Poyet, Cédric; Wagner, Ulrich; Guo, Tiannan; Aebersold, Ruedi; Garraway, Levi A; Wild, Peter J; Theurillat, Jean-Philippe; Brown, Myles

    2016-06-13

    Androgen receptor (AR) signaling is a key driver of prostate cancer (PC). While androgen-deprivation therapy is transiently effective in advanced disease, tumors often progress to a lethal castration-resistant state (CRPC). We show that recurrent PC-driver mutations in speckle-type POZ protein (SPOP) stabilize the TRIM24 protein, which promotes proliferation under low androgen conditions. TRIM24 augments AR signaling, and AR and TRIM24 co-activated genes are significantly upregulated in CRPC. Expression of TRIM24 protein increases from primary PC to CRPC, and both TRIM24 protein levels and the AR/TRIM24 gene signature predict disease recurrence. Analyses in CRPC cells reveal that the TRIM24 bromodomain and the AR-interacting motif are essential to support proliferation. These data provide a rationale for therapeutic TRIM24 targeting in SPOP mutant and CRPC patients.

  17. Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

    Science.gov (United States)

    Takito, Jiro; Kimura, Junko; Kajima, Koji; Uozumi, Nobuyuki; Watanabe, Makoto; Yokosuka, Akihito; Mimaki, Yoshihiro; Nakamura, Masanori; Ohizumi, Yasushi

    2016-07-01

    Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells. PMID:27128150

  18. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.

    Science.gov (United States)

    Dalton, Jutta C; Bätz, Ulrike; Liu, Jason; Curie, Gemma L; Quail, Peter H

    2016-01-01

    Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5'-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation. PMID:27379152

  19. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3.

    Science.gov (United States)

    Dalton, Jutta C; Bätz, Ulrike; Liu, Jason; Curie, Gemma L; Quail, Peter H

    2016-01-01

    Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5'-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation.

  20. A Modified Reverse One-Hybrid Screen Identifies Transcriptional Activation Domains in PHYTOCHROME-INTERACTING FACTOR 3

    Science.gov (United States)

    Dalton, Jutta C.; Bätz, Ulrike; Liu, Jason; Curie, Gemma L.; Quail, Peter H.

    2016-01-01

    Transcriptional activation domains (TADs) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput manner. A plant transcriptional activator, PIF3 (phytochrome interacting factor 3), was fused to the yeast GAL4-DNA-binding Domain (BD), driving expression of the URA3 (Orotidine 5′-phosphate decarboxylase) reporter, and used for negative selection on 5-fluroorotic acid (5FOA). Randomly mutagenized variants of PIF3 were then selected for a loss or reduction in transcriptional activation activity by survival on FOA. In the process, we developed a strategy to eliminate false positives from negative selection that can be used for both reverse-1- and 2-hybrid screens. With this method we were able to identify two distinct regions in PIF3 with transcriptional activation activity, both of which are functionally conserved in PIF1, PIF4, and PIF5. Both are collectively necessary for full PIF3 transcriptional activity, but neither is sufficient to induce transcription autonomously. We also found that the TAD appear to overlap physically with other PIF3 functions, such as phyB binding activity and consequent phosphorylation. Our protocol should provide a valuable tool for identifying, analyzing and characterizing novel TADs in eukaryotic transcription factors, and thus potentially contribute to the unraveling of the mechanism underlying transcriptional activation. PMID:27379152

  1. Role of SIRT1 and FOXO factors in eNOS transcriptional activation by resveratrol.

    Science.gov (United States)

    Xia, Ning; Strand, Susanne; Schlufter, Frank; Siuda, Daniel; Reifenberg, Gisela; Kleinert, Hartmut; Förstermann, Ulrich; Li, Huige

    2013-08-01

    Many of the cardiovascular protective effects of resveratrol are attributable to an enhanced production of nitric oxide (NO) by the endothelial NO synthase (eNOS). Resveratrol has been shown to enhance eNOS gene expression as well as eNOS enzymatic activity. The aim of the present study was to analyze the molecular mechanisms of eNOS transcriptional activation by resveratrol. Treatment of human EA.hy 926 endothelial cells with resveratrol led to a concentration-dependent upregulation of eNOS expression. In luciferase reporter gene assay, resveratrol enhanced the activity of human eNOS promoter fragments (3500, 1600, 633 and 263bp in length, respectively), indicating that the proximal promoter region is required for resveratrol-induced eNOS transcriptional activation. Knockdown of the NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1) by siRNA prevented the upregulation of eNOS mRNA and protein by resveratrol. Forkhead box O (FOXO) transcription factors are established downstream targets of SIRT1. siRNA-mediated knockdown of FOXO1 and FOXO3a abolished the effect of resveratrol on eNOS expression, indicating the involvement of these factors. Resveratrol treatment enhanced the expression of FOXO1 and FOXO3a in EA.hy 926 cells. Reporter gene assay using promoter containing forkhead response elements showed increased FOXO factor activity by resveratrol. In electrophoretic mobility shift assay, the enhanced binding of nuclear proteins to the eNOS promoter regions by resveratrol could be blocked by antibodies against FOXO1 and FOXO3a. In conclusion, resveratrol enhances the expression and activity of FOXO transcription factors. The SIRT1/FOXO factor axis is involved in resveratrol-induced eNOS transcriptional activation.

  2. Resveratrol inhibits growth of orthotopic pancreatic tumors through activation of FOXO transcription factors.

    Directory of Open Access Journals (Sweden)

    Sanjit K Roy

    Full Text Available BACKGROUND: The forkhead transcription factors of the O class (FOXO play a direct role in cellular proliferation, oxidative stress response, and tumorigenesis. The objectives of this study were to examine whether FOXOs regulate antitumor activities of resveratrol in pancreatic cancer cells in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Pancreatic cancer cell lines were treated with resveratrol. Cell viability, colony formation, apoptosis and cell cycle were measured by XTT, soft agar, TUNEL and flow cytometry assays, respectively. FOXO nuclear translocation, DNA binding and transcriptional activities were measured by fluorescence technique, gelshift and luciferase assay, respectively. Mice were orthotopically implanted with PANC1 cells and orally gavaged with resveratrol. The components of PI3K and ERK pathways, FOXOs and their target gene expressions were measured by the Western blot analysis. Resveratrol inhibited cell viability and colony formations, and induced apoptosis through caspase-3 activation in four pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Hs766T, and AsPC-1. Resveratrol induced cell cycle arrest by up-regulating the expression of p21/CIP1, p27/KIP1 and inhibiting the expression of cyclin D1. Resveratrol induced apoptosis by up-regulating Bim and activating caspase-3. Resveratrol inhibited phosphorylation of FOXOs, and enhanced their nuclear translocation, FOXO-DNA binding and transcriptional activities. The inhibition of PI3K/AKT and MEK/ERK pathways induced FOXO transcriptional activity and apoptosis. Furthermore, deletion of FOXO genes abrogated resveratrol-induced cell cycle arrest and apoptosis. Finally, resveratrol-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FOXO1 and FOXO3a, and induction of apoptosis and FOXO target genes. CONCLUSIONS: These data suggest that inhibition of ERK and AKT pathways act together to activate FOXO

  3. Genetic factors affecting gene transcription and catalytic activity of UDP-glucuronosyltransferases in human liver.

    Science.gov (United States)

    Liu, Wanqing; Ramírez, Jacqueline; Gamazon, Eric R; Mirkov, Snezana; Chen, Peixian; Wu, Kehua; Sun, Chang; Cox, Nancy J; Cook, Edwin; Das, Soma; Ratain, Mark J

    2014-10-15

    The aim of this study was to discover cis- and trans-acting factors significantly affecting mRNA expression and catalytic activity of human hepatic UDP-glucuronosyltransferases (UGTs). Transcription levels of five major hepatic UGT1A (UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9) and five UGT2B (UGT2B4, UGT2B7, UGT2B10, UGT2B15 and UGT2B17) genes were quantified in human liver tissue samples (n = 125) using real-time PCR. Glucuronidation activities of 14 substrates were measured in 47 livers. We genotyped 167 tagSNPs (single-nucleotide polymorphisms) in UGT1A (n = 43) and UGT2B (n = 124), as well as the known functional UGT1A1*28 and UGT2B17 CNV (copy number variation) polymorphisms. Transcription levels of 15 transcription factors (TFs) known to regulate these UGTs were quantified. We found that UGT expression and activity were highly variable among the livers (median and range of coefficient of variations: 135%, 74-217% and 52%, 39-105%, respectively). CAR, PXR and ESR1 were found to be the most important trans-regulators of UGT transcription (median and range of correlation coefficients: 46%, 6-58%; 47%, 9-58%; and 52%, 24-75%, respectively). Hepatic UGT activities were mainly determined by UGT gene transcription levels. Twenty-one polymorphisms were significantly (FDR-adjusted P transcription and testosterone glucuronidation rate, in addition to that attributable to the UGT2B17 CNV. Our study discovered novel pharmacogenetic markers and provided detailed insight into the genetic network regulating hepatic UGTs.

  4. Transcriptional activation of human CDCA8 gene regulated by transcription factor NF-Y in embryonic stem cells and cancer cells.

    Science.gov (United States)

    Dai, Can; Miao, Cong-Xiu; Xu, Xiao-Ming; Liu, Lv-Jun; Gu, Yi-Fan; Zhou, Di; Chen, Lian-Sheng; Lin, Ge; Lu, Guang-Xiu

    2015-09-11

    The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.

  5. Versatility of cooperative transcriptional activation: a thermodynamical modeling analysis for greater-than-additive and less-than-additive effects.

    Directory of Open Access Journals (Sweden)

    Till D Frank

    Full Text Available We derive a statistical model of transcriptional activation using equilibrium thermodynamics of chemical reactions. We examine to what extent this statistical model predicts synergy effects of cooperative activation of gene expression. We determine parameter domains in which greater-than-additive and less-than-additive effects are predicted for cooperative regulation by two activators. We show that the statistical approach can be used to identify different causes of synergistic greater-than-additive effects: nonlinearities of the thermostatistical transcriptional machinery and three-body interactions between RNA polymerase and two activators. In particular, our model-based analysis suggests that at low transcription factor concentrations cooperative activation cannot yield synergistic greater-than-additive effects, i.e., DNA transcription can only exhibit less-than-additive effects. Accordingly, transcriptional activity turns from synergistic greater-than-additive responses at relatively high transcription factor concentrations into less-than-additive responses at relatively low concentrations. In addition, two types of re-entrant phenomena are predicted. First, our analysis predicts that under particular circumstances transcriptional activity will feature a sequence of less-than-additive, greater-than-additive, and eventually less-than-additive effects when for fixed activator concentrations the regulatory impact of activators on the binding of RNA polymerase to the promoter increases from weak, to moderate, to strong. Second, for appropriate promoter conditions when activator concentrations are increased then the aforementioned re-entrant sequence of less-than-additive, greater-than-additive, and less-than-additive effects is predicted as well. Finally, our model-based analysis suggests that even for weak activators that individually induce only negligible increases in promoter activity, promoter activity can exhibit greater

  6. Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation.

    Science.gov (United States)

    Ngo, Huu B; Lovely, Geoffrey A; Phillips, Rob; Chan, David C

    2014-01-01

    TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)--the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

  7. An Efficient Method to Identify Conditionally Activated Transcription Factors and their Corresponding Signal Transduction Pathway Segments

    Directory of Open Access Journals (Sweden)

    Haiyan Hu

    2009-11-01

    Full Text Available A signal transduction pathway (STP is a cascade composed of a series of signal transferring steps, which often activate one or more transcription factors (TFs to control the transcription of target genes. Understanding signaling pathways is important to our understanding of the molecular mechanisms of disease. Many condition-annotated pathways have been deposited in public databases. However, condition-annotated pathways are far from complete, considering the large number of possible conditions. Computational methods to assist in the identification of conditionally activated pathways are greatly needed. In this paper, we propose an efficient method to identify conditionally activated pathway segments starting from the identification of conditionally activated TFs, by incorporating protein-DNA binding data, gene expression data and protein interaction data. Applying our methods on several microarray datasets, we have discovered many significantly activated TFs and their corresponding pathway segments, which are supported by evidence in the literature.

  8. Activation of transcriptional activity of HSE by a novel mouse zinc finger protein ZNFD specifically expressed in testis.

    Science.gov (United States)

    Xu, Fengqin; Wang, Weiping; Lei, Chen; Liu, Qingmei; Qiu, Hao; Muraleedharan, Vinaydhar; Zhou, Bin; Cheng, Hongxia; Huang, Zhongkai; Xu, Weian; Li, Bichun; Wang, Minghua

    2012-04-01

    Zinc finger proteins (ZFPs) that contain multiple cysteine and/or histidine residues perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation, and apoptosis. The Cys-Cys-His-His (C(2)H(2)) type of ZFPs are the well-defined members of this super family and are the largest and most complex proteins in eukaryotic genomes. In this study, we identified a novel C(2)H(2) type of zinc finger gene ZNFD from mice which has a 1,002 bp open reading frame and encodes a protein with 333 amino acid residues. The predicted 37.4 kDa protein contains a C(2)H(2) zinc finger domain. ZNFD gene is located on chromosome 18qD1. RT-PCR analysis revealed that the ZNFD gene was specifically expressed in mouse testis but not in other tissues. Subcellular localization analysis demonstrated that ZNFD was localized in the nucleus. Reporter gene assays showed that overexpression of ZNFD in the COS7 cells activates the transcriptional activities of heat shock element (HSE). Overall, these results suggest that ZNFD is a member of the zinc finger transcription factor family and it participates in the transcriptional regulation of HSE. Many heat shock proteins regulated by HSE are involved in testicular development. Therefore, our results suggest that ZNFD may probably participate in the development of mouse testis and function as a transcription activator in HSE-mediated gene expression and signaling pathways.

  9. Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein(RMP)

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase Ⅱ as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74of TFIIF subunits. In the immunoprecipitation assay in COS 1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-amino-acid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.

  10. Effect of nitrate on activities and transcript levels of nitrate reductase and glutamine synthetase in rice

    Institute of Scientific and Technical Information of China (English)

    CAO Yun; FAN Xiao-Rong; SUN Shu-Bin; XU Guo-Hua; HU Jiang; SHEN Qi-Rong

    2008-01-01

    Real-time polymerase chain reaction analysis was used to compare the effect of NO-3 on the activities of nitrate reductase (NR) and glutamine synthetase (GS),and the transcript levels of two NR genes,OsNia1 and OsNia2,two cytceolic GS1 genes,OsGln1;1 and OsGln1;2,and one plastid GS2 gene OsGln2,in two rice (Oryza sativa L.) cultivars Nanguang (NG) and Yunjing (YJ).Both cultivars achieved greater biomass and higher total N concentration when grown in a mixed N supply than in sole NH+ nutrition.Supply of NO-3 increased NR activity in both leaves and roots.Expression of both NR genes was also substantially enhanced and transcript levels of OsNia2 were significantly higher than those of OsNia1.NO-3 also caused an increase in GS activity,but had a complex effect on the expression of the three GS genes.In roots,the OsGln1;1 transcript increased,but OsGln1;2 decreased.In leaves,NO-3 had no effect on the GS1 expression,but the transcript for OsGln2 increased both in the leaves and roots of rice with a mixed supply of N.These results suggested that the increase in GS activity might be a result of the complicated regulation of the various GS genes.In addition,the NO-3 induced increase of biomass,NR activity,GS activity,and the transcript levels of NR and GS genes were proportionally higher in NG than in YJ,indicating a stronger response of NG to NO-3 nutrition than YJ.

  11. Comprehensive analysis of the specificity of transcription activator-like effector nucleases

    DEFF Research Database (Denmark)

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien;

    2014-01-01

    their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator...

  12. Modulation of CP2 family transcriptional activity by CRTR-1 and sumoylation.

    Directory of Open Access Journals (Sweden)

    Sarah To

    Full Text Available CRTR-1 is a member of the CP2 family of transcription factors. Unlike other members of the family which are widely expressed, CRTR-1 expression shows specific spatio-temporal regulation. Gene targeting demonstrates that CRTR-1 plays a central role in the maturation and function of the salivary glands and the kidney. CRTR-1 has also recently been identified as a component of the complex transcriptional network that maintains pluripotency in embryonic stem (ES cells. CRTR-1 was previously shown to be a repressor of transcription. We examine the activity of CRTR-1 in ES and other cells and show that CRTR-1 is generally an activator of transcription and that it modulates the activity of other family members, CP2, NF2d9 and altNF2d9, in a cell specific manner. We also demonstrate that CRTR-1 activity is regulated by sumoylation at a single major site, residue K30. These findings imply that functional redundancy with other family members may mask important roles for CRTR-1 in other tissues, including the blastocyst stage embryo and embryonic stem cells.

  13. IscR regulates RNase LS activity by repressing rnlA transcription.

    Science.gov (United States)

    Otsuka, Yuichi; Miki, Kumiko; Koga, Mitsunori; Katayama, Natsu; Morimoto, Wakako; Takahashi, Yasuhiro; Yonesaki, Tetsuro

    2010-07-01

    The Escherichia coli endoribonuclease LS was originally identified as a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. This RNase also plays an important role in RNA metabolisms in E. coli. rnlA is an essential gene for RNase LS activity, but the transcriptional regulation of this gene remains to be elucidated. An Fe-S cluster protein, IscR, acts as a transcription factor and controls the expression of genes that are necessary for Fe-S cluster biogenesis. Here, we report that overexpression of IscR suppressed RNase LS activity, causing the loss of antagonist activity against phage T4. This suppressive effect did not require the ligation of Fe-S cluster into IscR. beta-Galactosidase reporter assays showed that transcription from an rnlA promoter increased in iscR-deleted cells compared to wild-type cells, and gel-mobility shift assays revealed specific binding of IscR to the rnlA promoter region. RT-PCR analysis demonstrated that endogenous rnlA mRNA was reduced by overexpression of IscR and increased by deletion of iscR. From these results, we conclude that IscR negatively regulates transcription of rnlA and represses RNase LS activity.

  14. E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene

    DEFF Research Database (Denmark)

    Koziczak, M; Müller, H; Helin, K;

    2001-01-01

    -sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI-1 mRNA expression. In U2OS cells stably expressing an E2F1-estrogen receptor chimeric protein that could be activated by tamoxifen, PAI-1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide...

  15. Physiological and transcriptional responses of nitrifying bacteria exposed to copper in activated sludge.

    Science.gov (United States)

    Ouyang, Fan; Zhai, Hongyan; Ji, Min; Zhang, Hongyang; Dong, Zhao

    2016-01-15

    Cu inhibition of gene transcription in ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were rarely studied simultaneously in activated sludge. In this study, the transcription of amoA (for AOB) and nxrB (for NOB), nitrification efficiencies, AOB and NOB respiratory rates, and Cu distribution were simultaneously investigated. Modeling the relationships among the aforementioned parameters revealed that in complex activated sludge systems, nitrification efficiency was an insensitive parameter for showing Cu inhibition. Respiration activities and gene transcription were sensitive to Cu and positively correlated with each other. The transcription of amoA and nxrB genes indicated that the Cu had different inhibitory effects on AOB and NOB. AOB were more susceptible to Cu toxicity than NOB. Moreover, the degree of Cu inhibition on ammonia oxidation was greater than on nitrite oxidation. The analysis and related modeling results indicate that the inhibitory actions of Cu on nitrifying bacteria could mainly be attributed to intracellular Cu. The findings from this study provide insight into the mechanism of Cu inhibition on nitrification in complex activated sludge systems. PMID:26348150

  16. RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

    Science.gov (United States)

    Baranello, Laura; Wojtowicz, Damian; Cui, Kairong; Devaiah, Ballachanda N; Chung, Hye-Jung; Chan-Salis, Ka Yim; Guha, Rajarshi; Wilson, Kelli; Zhang, Xiaohu; Zhang, Hongliang; Piotrowski, Jason; Thomas, Craig J; Singer, Dinah S; Pugh, B Franklin; Pommier, Yves; Przytycka, Teresa M; Kouzine, Fedor; Lewis, Brian A; Zhao, Keji; Levens, David

    2016-04-01

    We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.

  17. Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter

    Directory of Open Access Journals (Sweden)

    Dan Liu

    2016-01-01

    Full Text Available HtrA serine peptidase 2 (HtrA2, also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at −1205~−838 bp and −146~+93 bp, with the −838~−649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at −709~+37 bp, and eight heat shock transcription factor 1 (HSF1 sites, two Sp1 transcription factor (SP1sites, one activator protein (AP site, seven p53 sites, and four YY1 transcription factor(YY1 sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases.

  18. Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA.

    Science.gov (United States)

    Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

    2016-07-01

    The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα.

  19. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray™ design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.

  20. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang-pil [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Chung, Sung Woon [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Hong, Ki Whan; Kim, Chi Dae [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  1. Transcriptional activation of peroxisome proliferator-activated receptor-γ requires activation of both protein kinase A and Akt during adipocyte differentiation

    International Nuclear Information System (INIS)

    Research highlights: → Elevated cAMP activates both PKA and Epac. → PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. → Akt modulates PPAR-γ transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

  2. A transcriptional activator is located in the coding region of the yeast PGK gene.

    OpenAIRE

    Mellor, J; Dobson, M J; Kingsman, A J; Kingsman, S M

    1987-01-01

    Expression of heterologous genes from the PGK promoter on high copy number plasmids in yeast is relatively poor compared to the intact PGK gene because of low steady-state RNA levels. In this paper we show that low levels of heterologous RNA are not due to instability of mRNA but result from inefficient transcription due to a defect in RNA synthesis. A comparison of RNA levels from homologous and heterologous transcription units allowed the identification of a positive activator for transcrip...

  3. Staf, a promiscuous activator for enhanced transcription by RNA polymerases II and III.

    OpenAIRE

    Schaub, M; Myslinski, E; Schuster, C.; Krol, A.; Carbon, P

    1997-01-01

    Staf is a zinc finger protein that we recently identified as the transcriptional activator of the RNA polymerase III-transcribed selenocysteine tRNA gene. In this work we demonstrate that enhanced transcription of the majority of vertebrate snRNA and snRNA-type genes, transcribed by RNA polymerases II and III, also requires Staf. DNA binding assays and microinjection of mutant genes into Xenopus oocytes showed the presence of Staf-responsive elements in the genes for human U4C, U6, Y4 and 7SK...

  4. Construction and Activity Assay of the Activating Transcription Factor 3 Reporter Vector pATF/CRE-luc

    Institute of Scientific and Technical Information of China (English)

    Jun-Qing XU; Jing-Lan DENG; You-Sheng WU; Han-Yan FU; Rui-Hua WANG; Jian ZHANG; Fan LU; Zhong-Liang ZHAO

    2006-01-01

    Activating transcription factor 3 (ATF3), a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors, is induced by many physiological stresses. To investigate the activity of ATF/CREB in cells with physiological stresses, we developed a practical reporter vector, the plasmid pATF/CRE-luc, bearing activating transcription factor/cAMP responsive element (ATF/CRE) binding sites. This plasmid was constructed by inserting three repeats of the ATF/CRE binding element into the plasmid pG51uc, replacing the GAL-4 binding sites. The plasmids pACT/ATF3 and pATF/CRE-luc were transfected into HeLa and NIH3T3 cells, respectively, and the results showed that the expression of luciferase was increased in a dose-dependent manner on plasmid pACT/ATF3. The data suggested that the plasmid pATF/CRE-luc could be used as a sensitive and convenient reporter system of ATF3 activity.

  5. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Benoît Couvigny

    Full Text Available The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor, we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

  6. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

    Science.gov (United States)

    Couvigny, Benoît; de Wouters, Tomas; Kaci, Ghalia; Jacouton, Elsa; Delorme, Christine; Doré, Joël; Renault, Pierre; Blottière, Hervé M; Guédon, Eric; Lapaque, Nicolas

    2015-01-01

    The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB) in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor), we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health. PMID:25946041

  7. Inhibition of p53 transcriptional activity by human cytomegalovirus UL44.

    Science.gov (United States)

    Kwon, Yejin; Kim, Mi-Na; Young Choi, Eun; Heon Kim, Jung; Hwang, Eung-Soo; Cha, Chang-Yong

    2012-05-01

    Human cytomegalovirus (HCMV) stimulates cellular synthesis of DNA and proteins and induces transition of the cell cycle from G(1) to S and G(2) /M phase, in spite of increased amounts of p53 in the infected cells. The immediate early protein IE2-86  kDa (IE86) tethers a transcriptional repression domain to p53; however, its repression of p53 function is not enough to abrogate the G(1) checkpoint function of p53. Other HCMV proteins that suppress the activity of p53 were investigated in this study. Of the HCMV proteins that bind to p53 when assessed by immunoprecipitation and immunoblot analysis, HCMV UL44 was chosen as a candidate protein. It was found that reporter gene containing p53 consensus sequence was activated by transfection with wild type p53, but when plasmids of p53 with IE86 or UL44 were co-transfected, p53 transcriptional activity was decreased to 3-7% of the p53 control in a dose-dependent manner. When the deletion mutant of UL44 was co-transected with p53, the carboxyl one-third portion of UL44 had little effect on inhibition of p53 transcriptional activity. The amount of mRNA p21 was measured in H1299 by real time PCR after transfection of the combination of p53 and UL44 vectors and it was found that p21 transcription by p53 was inhibited dose-dependently by UL44. Increased G0/G1 and decreased S phases in p53 wild type-transfected H1299 cells were recovered to the level of p53 mutant type-transfected ones by the additional transfection of UL44 in a dose-dependent manner. In conclusion, the transcriptional activity of p53 is suppressed by UL44 as well as by IE86. PMID:22376288

  8. A transcription activator-like effector (TALE) induction system mediated by proteolysis.

    Science.gov (United States)

    Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

    2016-04-01

    Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic. PMID:26854666

  9. Calmodulin-binding transcription activators and perspectives for applications in biotechnology.

    Science.gov (United States)

    Shen, Chenjia; Yang, Yanjun; Du, Liqun; Wang, Huizhong

    2015-12-01

    In recent years, a novel family of calmodulin-binding transcription activators (CAMTAs) has been reported in various species. The CAMTAs share a conserved domain organization, with a CG-1 DNA-binding domain, a transcription factor immunoglobulin domain, several ankyrin repeats, a calmodulin-binding domain, and a varying number of IQ motifs. CAMTAs participate in transcriptional regulation by recognizing and binding to a specific cis-element: (G/A/C)CGCG(C/G/T). Plants suffer from the environmental challenges, including abiotic and biotic stresses. Investigations in various plant species indicate a broad range of CAMTA functions involved in developmental regulation, environmental stress response, and hormone cross talk. In this review, we focus on the expression patterns and biological functions of CAMTAs to explore their probable applications in biotechnology. Furthermore, the identification and phylogenetic analysis of CAMTAs in crops could open new perspectives for enhancing stress tolerance, which could lead to improved crop production.

  10. Rph1 mediates the nutrient-limitation signaling pathway leading to transcriptional activation of autophagy.

    Science.gov (United States)

    Bernard, Amélie; Klionsky, Daniel J

    2015-04-01

    To maintain proper cellular homeostasis, the magnitude of autophagy activity has to be finely tuned in response to environmental changes. Many aspects of autophagy regulation have been extensively studied: pathways integrating signals through the master regulators TORC1 and PKA lead to multiple post-translational modifications affecting the functions, protein-protein interactions, and localization of Atg proteins. The expression of several ATG genes increases sharply upon autophagy induction conditions, and defects in ATG gene expression are associated with various diseases, pointing to the importance of transcriptional regulation of autophagy. Yet, how changes in ATG gene expression affect the rate of autophagy is not well characterized, and transcriptional regulators of the autophagy pathway remain largely unknown. To identify such regulators, we analyzed the expression of several ATG genes in a library of DNA-binding protein mutants. This led to the identification of Rph1 as a master transcriptional regulator of autophagy.

  11. Regulation and function of signal transducer and activator of transcription 3

    Institute of Scientific and Technical Information of China (English)

    Qian-Rong; Qi; Zeng-Ming; Yang

    2014-01-01

    Signal transducer and activator of transcription 3(STAT3), a member of the STAT family, is a key regulator of many physiological and pathological processes. Significant progress has been made in understanding the transcriptional control, posttranslational modification, cellular localization and functional regulation of STAT3. STAT3 can translocate into the nucleus and bind to specific promoter sequences, thereby exerting transcriptional regulation. Recent studies have shown that STAT3 can also translocate into mitochondria, participating in aerobic respiration and apoptosis. In addition, STAT3 plays an important role in inflammation and tumorigenesis by regulating cell proliferation, differentiation and metabolism. Conditional knockout mouse models make it possible to study the physiological function of STAT3 in specific tissues and organs. This review summarizes the latest advances in the understanding of the expression, regulation and function of STAT3 in physiological and tumorigenic processes.

  12. A transcription activator-like effector (TALE) induction system mediated by proteolysis.

    Science.gov (United States)

    Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

    2016-04-01

    Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

  13. Occludin controls HIV transcription in brain pericytes via regulation of SIRT-1 activation.

    Science.gov (United States)

    Castro, Victor; Bertrand, Luc; Luethen, Mareen; Dabrowski, Sebastian; Lombardi, Jorge; Morgan, Laura; Sharova, Natalia; Stevenson, Mario; Blasig, Ingolf E; Toborek, Michal

    2016-03-01

    HIV invades the brain early after infection; however, its interactions with the cells of the blood-brain barrier (BBB) remain poorly understood. Our goal was to evaluate the role of occludin, one of the tight junction proteins that regulate BBB functions in HIV infection of BBB pericytes. We provide evidence that occludin levels largely control the metabolic responses of human pericytes to HIV. Occludin in BBB pericytes decreased by 10% during the first 48 h after HIV infection, correlating with increased nuclear translocation of the gene repressor C-terminal-binding protein (CtBP)-1 and NFκB-p65 activation. These changes were associated with decreased expression and activation of the class III histone deacetylase sirtuin (SIRT)-1. Occludin levels recovered 96 h after infection, restoring SIRT-1 and reducing HIV transcription to 20% of its highest values. We characterized occludin biochemically as a novel NADH oxidase that controls the expression and activation of SIRT-1. The inverse correlation between occludin and HIV transcription was then replicated in human primary macrophages and differentiated monocytic U937 cells, in which occludin silencing resulted in 75 and 250% increased viral transcription, respectively. Our work shows that occludin has previously unsuspected metabolic properties and is a target of HIV infection, opening the possibility of designing novel pharmacological approaches to control HIV transcription.

  14. Occludin controls HIV transcription in brain pericytes via regulation of SIRT-1 activation.

    Science.gov (United States)

    Castro, Victor; Bertrand, Luc; Luethen, Mareen; Dabrowski, Sebastian; Lombardi, Jorge; Morgan, Laura; Sharova, Natalia; Stevenson, Mario; Blasig, Ingolf E; Toborek, Michal

    2016-03-01

    HIV invades the brain early after infection; however, its interactions with the cells of the blood-brain barrier (BBB) remain poorly understood. Our goal was to evaluate the role of occludin, one of the tight junction proteins that regulate BBB functions in HIV infection of BBB pericytes. We provide evidence that occludin levels largely control the metabolic responses of human pericytes to HIV. Occludin in BBB pericytes decreased by 10% during the first 48 h after HIV infection, correlating with increased nuclear translocation of the gene repressor C-terminal-binding protein (CtBP)-1 and NFκB-p65 activation. These changes were associated with decreased expression and activation of the class III histone deacetylase sirtuin (SIRT)-1. Occludin levels recovered 96 h after infection, restoring SIRT-1 and reducing HIV transcription to 20% of its highest values. We characterized occludin biochemically as a novel NADH oxidase that controls the expression and activation of SIRT-1. The inverse correlation between occludin and HIV transcription was then replicated in human primary macrophages and differentiated monocytic U937 cells, in which occludin silencing resulted in 75 and 250% increased viral transcription, respectively. Our work shows that occludin has previously unsuspected metabolic properties and is a target of HIV infection, opening the possibility of designing novel pharmacological approaches to control HIV transcription. PMID:26601824

  15. The transcription factor DBP affects circadian sleep consolidation and rhythmic EEG activity

    OpenAIRE

    Franken, Paulus; Lopez Molina, Luis; Marcacci, Lysiane; Schibler, Ulrich; Tafti, Mehdi

    2000-01-01

    Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues. Mice lacking DBP display a shorter circadian period in locomotor activity and are less active. Thus, although DBP is not essential for circadian rhythm generation, it does modulate important clock outputs. We studied the role of DBP in the circadian and homeosta...

  16. Structural and biochemical studies of sigma54 transcriptional activation in Aquifex aeolicus

    OpenAIRE

    Vidangos, Natasha Keith

    2010-01-01

    This thesis addresses a diversity of questions regarding the structural details of sigma54 transcriptional activation, and the function of sigma54 activation in the hyperthermophile Aquifex aeolicus. In order to place each topic in its appropriate context, a general introduction is provided in the first chapter, and supplemented with additional, more detailed introductions in each subsequent chapter. The second chapter reflects the central project of this thesis, the determination of the s...

  17. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    OpenAIRE

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Ping WANG; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for ea...

  18. A Transcriptional Mechanism Integrating Inputs from Extracellular Signals to Activate Hippocampal Stem Cells

    OpenAIRE

    Andersen, Jimena; Urbán, Noelia; Achimastou, Angeliki; Ito, Ayako; Simic, Milesa; Ullom, Kristy; Martynoga, Ben; Lebel, Mélanie; Göritz, Christian; Frisén, Jonas; Nakafuku, Masato; Guillemot, François

    2014-01-01

    Summary The activity of adult stem cells is regulated by signals emanating from the surrounding tissue. Many niche signals have been identified, but it is unclear how they influence the choice of stem cells to remain quiescent or divide. Here we show that when stem cells of the adult hippocampus receive activating signals, they first induce the expression of the transcription factor Ascl1 and only subsequently exit quiescence. Moreover, lowering Ascl1 expression reduces the proliferation rate...

  19. EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors

    OpenAIRE

    Florkowska Magdalena; Tymoszuk Piotr; Balwierz Aleksandra; Skucha Anna; Kochan Jakub; Wawro Mateusz; Stalinska Krystyna; Kasza Aneta

    2012-01-01

    Abstract Background Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by...

  20. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

    Science.gov (United States)

    Konermann, Silvana; Brigham, Mark D; Trevino, Alexandro E; Joung, Julia; Abudayyeh, Omar O; Barcena, Clea; Hsu, Patrick D; Habib, Naomi; Gootenberg, Jonathan S; Nishimasu, Hiroshi; Nureki, Osamu; Zhang, Feng

    2015-01-29

    Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

  1. PARP1 orchestrates variant histone exchange in signal-mediated transcriptional activation.

    Science.gov (United States)

    O'Donnell, Amanda; Yang, Shen-Hsi; Sharrocks, Andrew D

    2013-12-01

    Transcriptional activation is accompanied by multiple molecular events that remodel the local chromatin environment in promoter regions. These molecular events are often orchestrated in response to the activation of signalling pathways, as exemplified by the response of immediate early genes such as FOS to ERK MAP kinase signalling. Here, we demonstrate that inducible NFI recruitment permits PARP1 binding to the FOS promoter by a mutually reinforcing loop. PARP1 and its poly(ADP-ribosyl)ation activity are required for maintaining FOS activation kinetics. We also show that the histone variant H2A.Z associates with the FOS promoter and acts in a transcription-suppressive manner. However, in response to ERK pathway signalling, H2A.Z is replaced by H2A; PARP1 activity is required to promote this exchange. Thus, our work has revealed an additional facet of PARP1 function in promoting dynamic remodelling of promoter-associated nucleosomes to allow transcriptional activation in response to cellular signalling.

  2. Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

    Science.gov (United States)

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-02-18

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE. PMID:26748097

  3. Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

    Science.gov (United States)

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-02-18

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.

  4. Transcriptional activation of REST by Sp1 in Huntington's disease models.

    Directory of Open Access Journals (Sweden)

    Myriam Ravache

    Full Text Available In Huntington's disease (HD, mutant huntingtin (mHtt disrupts the normal transcriptional program of disease neurons by altering the function of several gene expression regulators such as Sp1. REST (Repressor Element-1 Silencing Transcription Factor, a key regulator of neuronal differentiation, is also aberrantly activated in HD by a mechanism that remains unclear. Here, we show that the level of REST mRNA is increased in HD mice and in NG108 cells differentiated into neuronal-like cells and expressing a toxic mHtt fragment. Using luciferase reporter gene assay, we delimited the REST promoter regions essential for mHtt-mediated REST upregulation and found that they contain Sp factor binding sites. We provide evidence that Sp1 and Sp3 bind REST promoter and interplay to fine-tune REST transcription. In undifferentiated NG108 cells, Sp1 and Sp3 have antagonistic effect, Sp1 acting as an activator and Sp3 as a repressor. Upon neuronal differentiation, we show that the amount and ratio of Sp1/Sp3 proteins decline, as does REST expression, and that the transcriptional role of Sp3 shifts toward a weak activator. Therefore, our results provide new molecular information to the transcriptional regulation of REST during neuronal differentiation. Importantly, specific knockdown of Sp1 abolishes REST upregulation in NG108 neuronal-like cells expressing mHtt. Our data together with earlier reports suggest that mHtt triggers a pathogenic cascade involving Sp1 activation, which leads to REST upregulation and repression of neuronal genes.

  5. CD studies of interaction of a bZIP oligopeptide model with DNA

    Science.gov (United States)

    Votavová, Hana; Točík, Zdeněk; Šponar, Jaroslav

    1999-05-01

    A leucine zipper (bZIP) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZIP proteins (Jun) with related DNA binding specificity. Selfcomplementary DNA hexadecanucleotides containing modified ATF/CRE target sites were used to study peptide-DNA complex formation. Four oligonucleotides contained substitutions of two GC or AT pairs by IC pairs in the ATF/CRE target sequence. In two other oligonucleotides there was a substitution of A by I in two AT pairs (mismatch IT pairs were presumably formed in the duplex) and one oligonucleotide contained I instead of C in two base pairs (IG mismatch in the duplex). Conformation changes of BP1 that occur on complex formation were studied by circular dichroism spectroscopy. The binding of peptide BP1 to oligonucleotides is accompanied by an increase of the α-helix content, which depends strongly on the oligonucleotide sequence. The substitution of two GC pairs within the specific binding site has either none or only a small effect. However, the substitution of two AT pairs within the binding site by IC strongly decreases the specificity of binding to a level observed with an oligonucleotide containing the C/EBP binding site, differing from the ATF/CRE site at four positions (Votavová et al., J. Biomol. Struct. Dyn. 3 (1997) 587). Similar results were obtained also with an oligonucleotide containing I instead of C in two base pairs (IG mismatch in the duplex). Two oligonucleotides with two substitutions of A by I but with unchanged T in the AT pairs (IT mismatch) showed smaller decrease in the α-helix formation on peptide binding than oligonucleotides, in which the whole AT pair was replaced by IC. The effect of such a substitution depends on the position of the original AT pairs in the target sequence, but the presence of T appears to be essential for specific peptide binding.

  6. Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants.

    Science.gov (United States)

    Czarnecka-Verner, Eva; Salem, Tarek A; Gurley, William B

    2016-02-01

    The Agrobacterium tumefaciens VirG response regulator of the VirA/VirG two-component system was adapted to function in tobacco protoplasts. The subcellular localization of VirG and VirA proteins transiently expressed in onion cells was determined using GFP fusions. Preliminary studies using Gal4DBD-VP16 fusions with VirG and Escherichia coli UhpA, and NarL response regulators indicated compatibility of these bacterial proteins with the eukaryotic transcriptional apparatus. A strong transcriptional activator based on tandem activation domains from the Drosophila fushi tarazu and Herpes simplex VP16 was created. Selected configurations of the two-site Gal4-vir box GUS reporters were activated by chimeric effectors dependent on either the yeast Gal4 DNA-binding domain or that of VirG. Transcriptional induction of the GUS reporter was highest for the VirE19-element promoter with both constitutive and wild-type VirG-tandem activation domain effectors. Multiple VirE19 elements increased the reporter activity proportionately, indicating that the VirG DNA binding domain was functional in plants. The VirG constitutive-Q-VP16 effector was more active than the VirG wild-type. In both the constitutive and wild-type forms of VirG, Q-VP16 activated transcription of the GUS reporter best when located at the C-terminus, i.e. juxtaposed to the VirG DNA binding domain. These results demonstrate the possibility of using DNA binding domains from bacterial response regulators and their cognate binding elements in the engineering of plant gene expression.

  7. Model of Transcriptional Activation By MarA in Escherichia Coli

    Science.gov (United States)

    Wall, Michael E.; Markowitz, David A.; Rosner, Judah L.; Martin, Robert G.

    2010-01-01

    We have developed a mathematical model of transcriptional activation by MarA in Escherichia coli, and used the model to analyze measurements of MarA-dependent activity of the marRAB, sodA, and micF promoters in mar-rob- cells. The model rationalizes an unexpected poor correlation between the mid-point of in vivo promoter activity profiles and in vitro equilibrium constants for MarA binding to promoter sequences. Analysis of the promoter activity data using the model yielded the following predictions regarding activation mechanisms: (1) MarA activation of the marRAB, sodA, and micF promoters involves a net acceleration of the kinetics of transitions after RNA polymerase binding, up to and including promoter escape and message elongation; (2) RNA polymerase binds to these promoters with nearly unit occupancy in the absence of MarA, making recruitment of polymerase an insignificant factor in activation of these promoters; and (3) instead of recruitment, activation of the micF promoter might involve a repulsion of polymerase combined with a large acceleration of the kinetics of polymerase activity. These predictions are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. A lack of recruitment in transcriptional activation represents an exception to the textbook description of activation of bacterial sigma-70 promoters. However, use of accelerated polymerase kinetics instead of recruitment might confer a competitive advantage to E. coli by decreasing latency in gene regulation.

  8. Reactive oxygen species in signalling the transcriptional activation of WIPK expression in tobacco.

    Science.gov (United States)

    Xu, Juan; Yang, Kwang-Yeol; Yoo, Seung Jin; Liu, Yidong; Ren, Dongtao; Zhang, Shuqun

    2014-07-01

    Plant mitogen-activated protein kinases represented by tobacco WIPK (wounding-induced protein kinase) and its orthologs in other species are unique in their regulation at transcriptional level in response to stress and pathogen infection. We previously demonstrated that transcriptional activation of WIPK is essential for induced WIPK activity, and activation of salicylic acid-induced protein kinase (SIPK) by the constitutively active NtMEK2(DD) is sufficient to induce WIPK gene expression. Here, we report that the effect of SIPK on WIPK gene expression is mediated by reactive oxygen species (ROS). Using a combination of pharmacological and gain-of-function transgenic approaches, we studied the relationship among SIPK activation, WIPK gene activation in response to fungal cryptogein, light-dependent ROS generation in chloroplasts, and ROS generated via NADPH oxidase. In the conditional gain-of-function GVG-NtMEK2(DD) transgenic tobacco, induction of WIPK expression is dependent on the ROS generation in chloroplasts. Consistently, methyl viologen, an inducer of ROS generation in chloroplasts, highly activated WIPK expression. In addition to chloroplast-originated ROS, H(2)O(2) generated from the cell-surface NADPH oxidase could also activate WIPK gene expression, and inhibition of cryptogein-induced ROS generation also abolished WIPK gene activation. Our data demonstrate that WIPK gene activation is mediated by ROS, which provides a mechanism by which ROS influence cellular signalling processes in plant stress/defence response.

  9. Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

    Science.gov (United States)

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E; Sha, Quan; Siebert, Jason; Gupta, Dave S; Schroeder, James W; Schleimer, Robert P

    2015-07-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  10. Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Qinghe Chen

    Full Text Available BACKGROUND: Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol. METHODOLOGY/PRINCIPAL FINDINGS: Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and

  11. Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

    Science.gov (United States)

    Thakore, Pratiksha I; Gersbach, Charles A

    2016-01-01

    Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy. PMID:26443215

  12. Functional domains of the transcriptional activator NUC-1 in Neurospora crassa.

    Science.gov (United States)

    Kang, S

    1993-08-25

    The NUC-1 regulatory protein directly controls the transcription of these genes and how the activity enzymes in Neurospora crassa. To understand how NUC-1 regulates the transcription of these genes and how the activity of NUC-1 is modulated by other regulatory proteins, two putative functional domains of NUC-1 were analysed: the DNA-binding domain and the regulatory domain. The DNA-binding activity of NUC-1 has not been directly demonstrated; however, results of deletion analysis, sequence analysis of the nuc-1 mutant alleles, and strong sequence similarity with the Saccharomyces cerevisiae PHO4 protein strongly suggest that the basic helix-loop-helix motif of NUC-1 forms a DNA-binding domain. Deletion and mutant analyses revealed that 39 amino acid (aa) residues (aa 463 to 501), or fewer, of NUC-1 are interacting with the negative regulatory factor(s), the PREG and/or PGOV proteins.

  13. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    International Nuclear Information System (INIS)

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-α transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling

  14. The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

    Science.gov (United States)

    Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

    2016-01-01

    The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

  15. Dimer formation and transcription activation in the sporulation response regulator Spo0A.

    Science.gov (United States)

    Lewis, Richard J; Scott, David J; Brannigan, James A; Ladds, Joanne C; Cervin, Marguerite A; Spiegelman, George B; Hoggett, James G; Barák, Imrich; Wilkinson, Anthony J

    2002-02-15

    The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A. PMID:11851334

  16. Wnt-induced transcriptional activation is exclusively mediated by TCF/LEF.

    Science.gov (United States)

    Schuijers, Jurian; Mokry, Michal; Hatzis, Pantelis; Cuppen, Edwin; Clevers, Hans

    2014-01-13

    Active canonical Wnt signaling results in recruitment of β-catenin to DNA by TCF/LEF family members, leading to transcriptional activation of TCF target genes. However, additional transcription factors have been suggested to recruit β-catenin and tether it to DNA. Here, we describe the genome-wide pattern of β-catenin DNA binding in murine intestinal epithelium, Wnt-responsive colorectal cancer (CRC) cells and HEK293 embryonic kidney cells. We identify two classes of β-catenin binding sites. The first class represents the majority of the DNA-bound β-catenin and co-localizes with TCF4, the prominent TCF/LEF family member in these cells. The second class consists of β-catenin binding sites that co-localize with a minimal amount of TCF4. The latter consists of lower affinity β-catenin binding events, does not drive transcription and often does not contain a consensus TCF binding motif. Surprisingly, a dominant-negative form of TCF4 abrogates the β-catenin/DNA interaction of both classes of binding sites, implying that the second class comprises low affinity TCF-DNA complexes. Our results indicate that β-catenin is tethered to chromatin overwhelmingly through the TCF/LEF transcription factors in these three systems.

  17. Notch-1 activates estrogen receptor-α-dependent transcription via IKKα in breast cancer cells

    Science.gov (United States)

    Hao, L; Rizzo, P; Osipo, C; Pannuti, A; Wyatt, D; Cheung, LW-K; Sonenshein, G; Osborne, BA; Miele, L

    2016-01-01

    Approximately 80% of breast cancers express the estrogen receptor-α (ERα) and are treated with anti-estrogens. Resistance to these agents is a major cause of mortality. We have shown that estrogen inhibits Notch, whereas anti-estrogens or estrogen withdrawal activate Notch signaling. Combined inhibition of Notch and estrogen signaling has synergistic effects in ERα-positive breast cancer models. However, the mechanisms whereby Notch-1 promotes the growth of ERα-positive breast cancer cells are unknown. Here, we demonstrate that Notch-1 increases the transcription of ERα-responsive genes in the presence or absence of estrogen via a novel chromatin crosstalk mechanism. Our data support a model in which Notch-1 can activate the transcription of ERα-target genes via IKKα-dependent cooperative chromatin recruitment of Notch–CSL–MAML1 transcriptional complexes (NTC) and ERα, which promotes the recruitment of p300. CSL binding elements frequently occur in close proximity to estrogen-responsive elements (EREs) in the human and mouse genomes. Our observations suggest that a hitherto unknown Notch-1/ERα chromatin crosstalk mediates Notch signaling effects in ERα-positive breast cancer cells and contributes to regulate the transcriptional functions of ERα itself. PMID:19838210

  18. Differential expression of two activating transcription factor 5 isoforms in papillary thyroid carcinoma

    Science.gov (United States)

    Vicari, Luisa; La Rosa, Cristina; Forte, Stefano; Calabrese, Giovanna; Colarossi, Cristina; Aiello, Eleonora; Salluzzo, Salvatore; Memeo, Lorenzo

    2016-01-01

    Background Activating transcription factor 5 (ATF5) is a member of the activating transcription/cAMP response element-binding protein family of basic leucine zipper proteins that plays an important role in cell survival, differentiation, proliferation, and apoptosis. The ATF5 gene generates two transcripts: ATF5 isoform 1 and ATF5 isoform 2. A number of studies indicate that ATF5 could be an attractive target for therapeutic intervention in several tumor types; however, so far, the role of ATF5 has not been investigated in papillary thyroid carcinoma (PTC). Methods Quantitative real-time reverse transcription polymerase chain reaction and immuno-histochemical staining were used to study ATF5 mRNA and protein expression in PTC. Results We report here that ATF5 is expressed more in PTC tissue than in normal thyroid tissue. Furthermore, this is the first study that describes the presence of both ATF5 isoforms in PTC. Conclusion These findings could provide potential applications in PTC cancer treatment.

  19. SUMOylation of the inducible (c-Fos:c-Jun)/AP-1 transcription complex occurs on target promoters to limit transcriptional activation.

    Science.gov (United States)

    Tempé, D; Vives, E; Brockly, F; Brooks, H; De Rossi, S; Piechaczyk, M; Bossis, G

    2014-02-13

    The inducible proto-oncogenic (c-Fos:c-Jun)/AP-1 transcription complex binds 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) in its target genes. It is tightly controlled at multiple levels to avoid the deleterious effects of its inappropriate activation. In particular, SUMOylation represses its transactivation capacity in transient reporter assays using constitutively expressed proteins. This led to the presumption that (c-Fos:c-Jun)/AP-1 SUMOylation would be required to turn-off transcription of its target genes, as proposed for various transcription factors. Instead, thanks to the generation of an antibody specific for SUMO-modified c-Fos, we provide here direct evidence that SUMOylated c-Fos is present on a stably integrated reporter TPA-inducible promoter at the onset of transcriptional activation and colocalizes with RNA polymerase II within chromatin. Interestingly, (c-Fos:c-Jun)/AP-1 SUMOylation limits reporter gene induction, as well as the appearance of active transcription-specific histone marks on its promoter. Moreover, non-SUMOylatable mutant (c-Fos:c-Jun)/AP-1 dimers accumulate to higher levels on their target promoter, suggesting that SUMOylation might facilitate the release of (c-Fos:c-Jun)/AP-1 from promoters. Finally, activation of GADD153, an AP-1 target gene, is also associated with a rapid increase in SUMOylation at the level of its TRE and c-Fos SUMOylation dampens its induction by TPA. Taken together, our data suggest that SUMOylation could serve to buffer transcriptional activation of AP-1 target genes.

  20. Benzimidazoles diminish ERE transcriptional activity and cell growth in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Payton-Stewart, Florastina [Department of Chemistry, College of Arts and Sciences, Xavier University of Louisiana, New Orleans, LA (United States); Tilghman, Syreeta L. [Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA (United States); Williams, LaKeisha G. [Division of Clinical and Administrative Sciences, College of Pharmacy Xavier University of Louisiana, New Orleans, LA (United States); Winfield, Leyte L., E-mail: lwinfield@spelman.edu [Department of Chemistry, Spelman College, Atlanta, GA (United States)

    2014-08-08

    Highlights: • The methyl-substituted benzimidazole was more effective at inhibiting growth in MDA-MB 231 cells. • The naphthyl-substituted benzimidazole was more effective at inhibiting growth in MCF-7 cells than ICI. • The benzimidazole molecules demonstrated a dose-dependent reduction in ERE transcriptional activity. • The benzimidazole molecules had binding mode in ERα and ERβ comparable to that of the co-crystallized ligand. - Abstract: Estrogen receptors (ERα and ERβ) are members of the nuclear receptor superfamily. They regulate the transcription of estrogen-responsive genes and mediate numerous estrogen related diseases (i.e., fertility, osteoporosis, cancer, etc.). As such, ERs are potentially useful targets for developing therapies and diagnostic tools for hormonally responsive human breast cancers. In this work, two benzimidazole-based sulfonamides originally designed to reduce proliferation in prostate cancer, have been evaluated for their ability to modulate growth in estrogen dependent and independent cell lines (MCF-7 and MDA-MB 231) using cell viability assays. The molecules reduced growth in MCF-7 cells, but differed in their impact on the growth of MDA-MB 231 cells. Although both molecules reduced estrogen response element (ERE) transcriptional activity in a dose dependent manner, the contrasting activity in the MDA-MB-231 cells seems to suggest that the molecules may act through alternate ER-mediated pathways. Further, the methyl analog showed modest selectivity for the ERβ receptor in an ER gene expression array panel, while the naphthyl analog did not significantly alter gene expression. The molecules were docked in the ligand binding domains of the ERα-antagonist and ERβ-agonist crystal structures to evaluate the potential of the molecules to interact with the receptors. The computational analysis complimented the results obtained in the assay of transcriptional activity and gene expression suggesting that the molecules

  1. P53 and p73 differ in their ability to inhibit glucocorticoid receptor (GR transcriptional activity

    Directory of Open Access Journals (Sweden)

    Nie Linghu

    2006-12-01

    Full Text Available Abstract Background p53 is a tumor suppressor and potent inhibitor of cell growth. P73 is highly similar to p53 at both the amino acid sequence and structural levels. Given their similarities, it is important to determine whether p53 and p73 function in similar or distinct pathways. There is abundant evidence for negative cross-talk between glucocorticoid receptor (GR and p53. Neither physical nor functional interactions between GR and p73 have been reported. In this study, we examined the ability of p53 and p73 to interact with and inhibit GR transcriptional activity. Results We show that both p53 and p73 can bind GR, and that p53 and p73-mediated transcriptional activity is inhibited by GR co-expression. Wild-type p53 efficiently inhibited GR transcriptional activity in cells expressing both proteins. Surprisingly, however, p73 was either unable to efficiently inhibit GR, or increased GR activity slightly. To examine the basis for this difference, a series of p53:p73 chimeric proteins were generated in which corresponding regions of either protein have been swapped. Replacing N- and C-terminal sequences in p53 with the corresponding sequences from p73 prevented it from inhibiting GR. In contrast, replacing p73 N- and C-terminal sequences with the corresponding sequences from p53 allowed it to efficiently inhibit GR. Differences in GR inhibition were not related to differences in transcriptional activity of the p53:p73 chimeras or their ability to bind GR. Conclusion Our results indicate that both N- and C-terminal regions of p53 and p73 contribute to their regulation of GR. The differential ability of p53 and p73 to inhibit GR is due, in part, to differences in their N-terminal and C-terminal sequences.

  2. p55PIK Transcriptionally Activated by MZF1 Promotes Colorectal Cancer Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Yu Deng

    2013-01-01

    Full Text Available p55PIK, regulatory subunit of class IA phosphatidylinositol 3-kinase (PI3K, plays a crucial role in cell cycle progression by interaction with tumor repressor retinoblastoma (Rb protein. A recent study showed that Rb protein can localize to the mitochondria in proliferative cells. Aberrant p55PIK expression may contribute to mitochondrial dysfunction in cancer progression. To reveal the mechanisms of p55PIK transcriptional regulation, the p55PIK promoter characteristics were analyzed. The data show that myeloid zinc finger 1, MZF1, is necessary for p55PIK gene transcription activation. ChIP (Chromatin immuno-precipitation assay shows that MZF1 binds to the cis-element “TGGGGA” in p55PIK promoter. In MZF1 overexpressed cells, the promoter activity, expression of p55PIK, and cell proliferation rate were observed to be significantly enhanced. Whereas in MZF1-silenced cells, the promoter activity and expression of p55PIK and cell proliferation level was statistically decreased. In CRC tissues, MZF1 and p55PIK mRNA expression were increased (P=0.046, P=0.047, resp.. A strong positive correlation (Rs=0.94 between MZF1 and p55PIK mRNA expression was observed. Taken together, we concluded that p55PIK is transcriptionally activated by MZF1, resulting in increased proliferation of colorectal cancer cells.

  3. DNA recognition by a σ(54) transcriptional activator from Aquifex aeolicus.

    Science.gov (United States)

    Vidangos, Natasha K; Heideker, Johanna; Lyubimov, Artem; Lamers, Meindert; Huo, Yixin; Pelton, Jeffrey G; Ton, Jimmy; Gralla, Jay; Berger, James; Wemmer, David E

    2014-10-23

    Transcription initiation by bacterial σ(54)-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain (DBD). The structurally characterized DBDs from activators all belong to the Fis (factor for inversion stimulation) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DBD of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ(54) activators. Two NtrC4-binding sites were identified upstream (-145 and -85bp) from the start of the lpxC gene, which is responsible for the first committed step in lipid A biosynthesis. This is the first experimental evidence for σ(54) regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145-binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homolog, Fis. The greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base-specific contacts contributing to affinity than for Fis. PMID:25158097

  4. Uncoupling RARA transcriptional activation and degradation clarifies the bases for APL response to therapies.

    Science.gov (United States)

    Ablain, Julien; Leiva, Magdalena; Peres, Laurent; Fonsart, Julien; Anthony, Elodie; de Thé, Hugues

    2013-04-01

    In PML/RARA-driven acute promyelocytic leukemia (APL), retinoic acid (RA) induces leukemia cell differentiation and transiently clears the disease. Molecularly, RA activates PML/RARA-dependent transcription and also initiates its proteasome-mediated degradation. In contrast, arsenic, the other potent anti-APL therapy, only induces PML/RARA degradation by specifically targeting its PML moiety. The respective contributions of RA-triggered transcriptional activation and proteolysis to clinical response remain disputed. Here, we identify synthetic retinoids that potently activate RARA- or PML/RARA-dependent transcription, but fail to down-regulate RARA or PML/RARA protein levels. Similar to RA, these uncoupled retinoids elicit terminal differentiation, but unexpectedly fail to impair leukemia-initiating activity of PML/RARA-transformed cells ex vivo or in vivo. Accordingly, the survival benefit conferred by uncoupled retinoids in APL mice is dramatically lower than the one provided by RA. Differentiated APL blasts sorted from uncoupled retinoid-treated mice retain PML/RARA expression and reinitiate APL in secondary transplants. Thus, differentiation is insufficient for APL eradication, whereas PML/RARA loss is essential. These observations unify the modes of action of RA and arsenic and shed light on the potency of their combination in mice or patients.

  5. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

    Directory of Open Access Journals (Sweden)

    José Perdomo

    Full Text Available Friend of GATA 2 (FOG-2, a co-factor of several GATA transcription factors (GATA-4, -5 and 6, is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K324, 471, 915, 955 [corrected]. Three of these residues are part of the characteristic SUMO consensus site (ψKXE, while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2's nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status.

  6. Effects of a methanolic fraction of soybean seeds on the transcriptional activity of peroxisome proliferator-activated receptors (PPAR

    Directory of Open Access Journals (Sweden)

    V.S. Carrara

    2009-06-01

    Full Text Available Since the anti-inflammatory, antidiabetic and hypolipidemic effects of soy isoflavones may be mediated by activation of peroxisome proliferator-activated receptors (PPAR, the present study investigated whether the methanolic fractions obtained from soybean seeds (E1 and soybean seed coats with hypocotyls (E2 could influence PPARα, PPARγ and PPARβ/δ transcriptional activity. The isoflavones from E1 and E2 were quantified by HPLC analysis. E1 and E2 were rich in isoflavones (daidzin, glycitin, genistin, malonyldaidzin, malonylglycitin, malonylgenistin, daidzein, glycitein, and genistein. Moreover, E1 and E2 showed no evidence of genetically modified material containing the gene CP4 EPSPS. To investigate PPAR transcriptional activity, human promonocytic U-937 cells were treated with E1 and E2 (200, 400, 800, and 1600 µg/mL, positive controls or vehicle. Data are reported as fold-activation of the luciferase reporter driven by the PPAR-responsive element. Dose-response analysis revealed that E1 and E2 induced the transcriptional activity of PPARα (P < 0.001, with activation comparable to that obtained with 0.1 mM bezafibrate (positive control at 1600 µg/mL (4-fold and 800 µg/mL (9-fold, respectively. In addition, dose-response analysis revealed that E1 and E2 activated PPARβ/δ (P < 0.05, and the activation at 800 µg/mL (4- and 9-fold, respectively was comparable to that of 0.1 mM bezafibrate (positive control. However, no effect on PPARγ was observed. Activation of PPARα is consistent with the lipid-lowering activity of soy isoflavones in vivo, but further studies are needed to determine the physiological significance of PPARβ/δ activation.

  7. Coordinating Cell Cycle Remodeling with Transcriptional Activation at the Drosophila MBT.

    Science.gov (United States)

    Blythe, Shelby A; Wieschaus, Eric F

    2015-01-01

    During the maternal-to-zygotic transition (MZT), major changes in cell cycle regulation coincide with large-scale zygotic genome activation. In this chapter, we discuss the current understanding of how the cell cycle is remodeled over the course of the Drosophila MZT, and how the temporal precision of this event is linked to contemporaneous alterations in genome-wide chromatin structure and transcriptional activity. The cell cycle is initially lengthened during the MZT by activation of the DNA replication checkpoint but, subsequently, zygotically supplied factors are essential for establishing lasting modifications to the cell cycle. PMID:26358872

  8. Transcriptional activity of human endogenous retrovirus in Albanian children with autism spectrum disorders.

    Science.gov (United States)

    Balestrieri, Emanuela; Cipriani, Chiara; Matteucci, Claudia; Capodicasa, Natale; Pilika, Anita; Korca, Ina; Sorrentino, Roberta; Argaw-Denboba, Ayele; Bucci, Ilaria; Miele, Martino Tony; Coniglio, Antonella; Alessandrelli, Riccardo; Sinibaldi Vallebona, Paola

    2016-09-01

    Recent studies suggest that autism spectrum disorders (ASD) result from interactions between genetic and environmental factors, whose possible links could be represented by epigenetic mechanisms. Here, we investigated the transcriptional activity of three human endogenous retrovirus (HERV) families, in peripheral blood mononuclear cells (PBMCs) from Albanian ASD children, by quantitative real-time PCR. We aimed to confirm the different expression profile already found in Italian ASD children, and to highlight any social and family health condition emerging from information gathered through a questionnaire, to be included among environmental risk factors. The presence of increased HERV-H transcriptional activity in all autistic patients could be understood as a constant epigenetic imprinting of the disease, potentially useful for early diagnosis and for the development of effective novel therapeutic strategies. PMID:27602423

  9. Using in vivo electroporation to identify hepatic LDL receptor promoter elements and transcription factors mediating activation of transcription by T3

    Directory of Open Access Journals (Sweden)

    Dayami Lopez

    2012-12-01

    Full Text Available The technique of in vivo electroporation was adapted to investigate the promoter elements and transcription factors mediating the rapid induction of hepatic LDL receptor expression in response to thyroid hormone. Direct comparisons between wild type and mutant promoter constructs were made within the same animal. It was demonstrated that both TREs at bp −612 and −156 were required for the l-triiodothyronine (T3 response. ChIP analysis showed that binding of TRβ1 to the −612 and −156 TREs was markedly stimulated by T3 in vivo. Introduction of siRNAs against TRβ1/RXRα with LDL receptor promoter-luciferase construct by in vivo electroporation demonstrated that these transcription factors play the major physiological role in the activation of hepatic LDL receptor transcription. The findings agree with those made by transfecting H4IIE cells in vitro thus validating this technique for in vivo studies of mechanisms of transcriptional regulation. The findings reported herein also indicated, for the first time, that PPARα and USF-2 were required for maximum transcriptional activation of the LDL receptor in response to T3 treatment.

  10. Structure, function, and tethering of DNA-binding domains in σ⁵⁴ transcriptional activators.

    Science.gov (United States)

    Vidangos, Natasha; Maris, Ann E; Young, Anisa; Hong, Eunmi; Pelton, Jeffrey G; Batchelor, Joseph D; Wemmer, David E

    2013-12-01

    We compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ(54) transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ(54)-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-like homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly. PMID:23818155

  11. FATS is a transcriptional target of p53 and associated with antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhang Xifeng

    2010-09-01

    Full Text Available Abstract Frequent mutations of p53 in human cancers exemplify its crucial role as a tumor suppressor transcription factor, and p21, a transcriptional target of p53, plays a central role in surveillance of cell-cycle checkpoints. Our previous study has shown that FATS stabilize p21 to preserve genome integrity. In this study we identified a novel transcript variant of FATS (GenBank: GQ499374 through screening a cDNA library from mouse testis, which uncovered the promoter region of mouse FATS. Mouse FATS was highly expressed in testis. The p53-responsive elements existed in proximal region of both mouse and human FATS promoters. Functional study indicated that the transcription of FATS gene was activated by p53, whereas such effect was abolished by site-directed mutagenesis in the p53-RE of FATS promoter. Furthermore, the expression of FATS increased upon DNA damage in a p53-dependent manner. FATS expression was silent or downregulated in human cancers, and overexpression of FATS suppressed tumorigenicity in vivo independently of p53. Our results reveal FATS as a p53-regulated gene to monitor genomic stability.

  12. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    Science.gov (United States)

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  13. Transcriptional activation of Epstein-Barr virus BRLF1 by USF1 and Rta.

    Science.gov (United States)

    Hung, Chen-Chia; Kuo, Chung-Wen; Wang, Wen-Hung; Chang, Tzu-Hsuan; Chang, Pey-Jium; Chang, Li-Kwan; Liu, Shih-Tung

    2015-09-01

    During its lytic cycle, Epstein-Barr virus (EBV) expresses Rta, a factor encoded by BRLF1 that activates the transcription of viral lytic genes. We found that upstream stimulating factor (USF) binds to E1, one of the five E boxes located at - 79 in the BRLF1 promoter (Rp), to activate BRLF1 transcription. Furthermore, Rta was shown to interact with USF1 in coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays, and confocal laser-scanning microscopy further confirmed that these two proteins colocalize in the nucleus. Rta was also found to bind with the E1 sequence in a biotin-labelled E1 probe, but only in the presence of USF1, suggesting that these two proteins likely form a complex on E1. We subsequently constructed p188mSZ, a reporter plasmid that contained the sequence from - 188 to +5 in Rp, within which the Sp1 site and Zta response element were mutated. In EBV-negative Akata cells cotransfected with p188mSZ and plasmids expressing USF1 and Rta, synergistic activation of Rp transcription was observed. However, after mutating the E1 sequence in p188mSZ, USF1 and Rta were no longer able to transactivate Rp, indicating that Rta autoregulates BRLF1 transcription via its interaction with USF1 on E1. This study showed that pUSF1 transfection after EBV lytic induction in P3HR1 cells increases Rta expression, indicating that USF1 activates Rta expression after the virus enters the lytic cycle. Together, these results reveal a novel mechanism by which USF interacts with Rta to promote viral lytic development, and provide additional insight into the viral-host interactions of EBV.

  14. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation.

    Science.gov (United States)

    Chen, Shaoyong; Gulla, Sarah; Cai, Changmeng; Balk, Steven P

    2012-03-01

    Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

  15. The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition

    OpenAIRE

    Bloodgood, Brenda L.; Sharma, Nikhil; Browne, Heidi Adlman; Trepman, Alissa Z.; Greenberg, Michael E.

    2013-01-01

    A heterogeneous population of inhibitory neurons controls the flow of information through a neural circuit1–3. Inhibitory synapses that form on pyramidal neuron dendrites modulate the summation of excitatory synaptic potentials4–6 and prevent the generation of dendritic calcium spikes7,8. Precisely timed somatic inhibition limits both the number of action potentials and the time window during which firing can occur8,9. The activity-dependent transcription factor NPAS4 regulates inhibitory syn...

  16. Activating transcription factor 3 is a negative regulator of allergic pulmonary inflammation

    OpenAIRE

    Gilchrist, Mark; Henderson, William R.; Clark, April E.; Simmons, Randi M.; Ye, Xin; Smith, Kelly D.; Aderem, Alan

    2008-01-01

    We recently demonstrated the pivotal role of the transcription factor (TF) activating TF 3 (ATF3) in dampening inflammation. We demonstrate that ATF3 also ameliorates allergen-induced airway inflammation and hyperresponsiveness in a mouse model of human asthma. ATF3 expression was increased in the lungs of mice challenged with ovalbumin allergen, and this was associated with its recruitment to the promoters of genes encoding Th2-associated cytokines. ATF3-deficient mice developed significantl...

  17. Cooperative activation of transcription by bovine papillomavirus type 1 E2 can occur over a large distance.

    OpenAIRE

    Thierry, F; Dostatni, N; Arnos, F; Yaniv, M

    1990-01-01

    The viral transcriptional factors encoded by the E2 open reading frame bind to the specific DNA sequence elements ACCGNNNNCGGT, allowing activation or repression of transcription. We have analyzed bovine papillomavirus type 1 E2 transactivation using recombinant genes containing E2-binding sites inserted at either 3' or 5' positions relative to the heterologous transcriptional initiation site of the herpes simplex virus thymidine kinase gene. In these hybrid plasmids, strong transactivation r...

  18. 24. The transcription factors and the relevant signaling pathways activated by low concentration MNNG

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Aims: To explore the transcription factors and related signal transduction pathways activated in the alkylating agents N-methyl-N'-nitro-N-nitrosoguanindine (MNNG) exposed cells which may involved in the mechanism of MNNG induced changes of gene expression, especially the elevation of DNA polymerase β expression and also the consequence of JNK kinase activation which were reported previously in this lab. Methods: Clontech Mercury pathway profiling system containing 8 different vectors in which a specific response element is located upstream from the SEAP-reporter gene were employed to detect the transcription factor activation in Vero cells treated with 0.2 μmol/L MNNG for 2 hours. Thoroughly, CREB phosphorylation, protein kinase A (PKA) and the cellular cAMP content were also assayed with PhosphoPlus CREB (ser-133) antibody kit, protein kinase assay kit and cAMP RIA kit respectively. Results: Among 8 different response elements, the expression of the reporter gene governed by the transcription factors CREB (cAMP response element binding protein), AP1 (activator protein 1), NF-κB (nuclear factor κ B) were elevated by 1.3, 1.4 and 1.3 times higber than control respectively. The level of activated CREB by Ser-133 phosphorylation was 2.08 times higher than control in cells treated with MNNG for 60 min, as measured by immunoblotting. The activity of CREB upstream kinase protein kinase A (PKA), which can phosphorylate CREB on ser-133 was also activated, and the activation peaked at 60 min (11.03±2.80 arbitrary units vs 0.86±0.43 of control). Also, cAMP levels were significantly raised after 60-minute-treatment, 1.52 times higher vs those in solvent control. Conclusion: In addition of previously reported JNK activation, we show here that low concentration alkylating agent MNNG can also activate the cAMP-PKA and NF-κB pathway. These in consequence induce the activation of transcription factors APl, CREB and NF-κB, which may related to the MNNG induced changes in

  19. Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

    Science.gov (United States)

    Winokur, P L; McBride, A A

    1992-11-01

    Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these domains are highly conserved among different papillomaviruses. An internal hinge region separates the two functional domains. The region varies in amino acid sequence and length among the E2 proteins of different papillomaviruses. A series of mutations were constructed within the E2 open reading frame which delete various regions of the conserved DNA binding and transactivation domains as well as the internal hinge region. Two mutated E2 proteins that lack portions of the conserved DNA-binding domain but which support DNA replication were identified using transient replication assays. These mutated E2 proteins were unable to function as transcriptional activators. Conversely, two E2 proteins containing large deletions of the hinge region were able to activate transcription, but were defective for replication. Thus, the replication and transactivation functions of the E2 protein are separable. PMID:1327758

  20. HSF1 transcriptional activity mediates alcohol induction of Vamp2 expression and GABA release

    Directory of Open Access Journals (Sweden)

    Florence P. Varodayan

    2013-12-01

    Full Text Available Many central synapses are highly sensitive to alcohol, and it is now accepted that short-term alterations in synaptic function may lead to longer term changes in circuit function. The regulation of postsynaptic receptors by alcohol has been well studied, but the mechanisms underlying the effects of alcohol on the presynaptic terminal are relatively unexplored. To identify a pathway by which alcohol regulates neurotransmitter release, we recently investigated the mechanism by which ethanol induces the Vamp2 gene, but not Vamp1, in mouse primary cortical cultures. These two genes encode isoforms of synaptobrevin, a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE protein required for synaptic vesicle fusion. We found that alcohol activates the transcription factor heat shock factor 1 (HSF1 to induce Vamp2 gene expression, while Vamp1 mRNA levels remain unaffected. As the Vamp2 gene encodes a SNARE protein, we then investigated whether ethanol exposure and HSF1 transcriptional activity alter neurotransmitter release using electrophysiology. We found that alcohol increased the frequency of γ-aminobutyric acid (GABA-mediated miniature IPSCs via HSF1, but had no effect on mEPSCs. Overall, these data indicate that alcohol induces HSF1 transcriptional activity to trigger a specific coordinated adaptation in GABAergic presynaptic terminals. This mechanism could explain some of the changes in synaptic function that occur soon after alcohol exposure, and may underlie some of the more enduring effects of chronic alcohol intake on local circuit function.

  1. The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28

    Science.gov (United States)

    Murphy, Kristin E.; Shylo, Natalia A.; Alexander, Katherine A.; Churchill, Angela J.; Copperman, Cecilia; García-García, María J.

    2016-01-01

    KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity. PMID:27658112

  2. Enhanced NFκB and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer

    International Nuclear Information System (INIS)

    Signaling pathways that converge on two different transcription factor complexes, NFκB and AP-1, have been identified in estrogen receptor (ER)-positive breast cancers resistant to the antiestrogen, tamoxifen. Two cell line models of tamoxifen-resistant ER-positive breast cancer, MCF7/HER2 and BT474, showing increased AP-1 and NFκB DNA-binding and transcriptional activities, were studied to compare tamoxifen effects on NFκB and AP-1 regulated reporter genes relative to tamoxifen-sensitive MCF7 cells. The model cell lines were treated with the IKK inhibitor parthenolide (PA) or the proteasome inhibitor bortezomib (PS341), alone and in combination with tamoxifen. Expression microarray data available from 54 UCSF node-negative ER-positive breast cancer cases with known clinical outcome were used to search for potential genes signifying upregulated NFκB and AP-1 transcriptional activity in association with tamoxifen resistance. The association of these genes with patient outcome was further evaluated using node-negative ER-positive breast cancer cases identified from three other published data sets (Rotterdam, n = 209; Amsterdam, n = 68; Basel, n = 108), each having different patient age and adjuvant tamoxifen treatment characteristics. Doses of parthenolide and bortezomib capable of sensitizing the two endocrine resistant breast cancer models to tamoxifen were capable of suppressing NFκB and AP-1 regulated gene expression in combination with tamoxifen and also increased ER recruitment of the transcriptional co-repressor, NCoR. Transcript profiles from the UCSF breast cancer cases revealed three NFκB and AP-1 upregulated genes – cyclin D1, uPA and VEGF – capable of dichotomizing node-negative ER-positive cases into early and late relapsing subsets despite adjuvant tamoxfien therapy and most prognostic for younger age cases. Across the four independent sets of node-negative ER-positive breast cancer cases (UCSF, Rotterdam, Amsterdam, Basel), high expression of

  3. Enhanced NFκB and AP-1 transcriptional activity associated with antiestrogen resistant breast cancer

    Directory of Open Access Journals (Sweden)

    Moore Dan H

    2007-04-01

    Full Text Available Abstract Background Signaling pathways that converge on two different transcription factor complexes, NFκB and AP-1, have been identified in estrogen receptor (ER-positive breast cancers resistant to the antiestrogen, tamoxifen. Methods Two cell line models of tamoxifen-resistant ER-positive breast cancer, MCF7/HER2 and BT474, showing increased AP-1 and NFκB DNA-binding and transcriptional activities, were studied to compare tamoxifen effects on NFκB and AP-1 regulated reporter genes relative to tamoxifen-sensitive MCF7 cells. The model cell lines were treated with the IKK inhibitor parthenolide (PA or the proteasome inhibitor bortezomib (PS341, alone and in combination with tamoxifen. Expression microarray data available from 54 UCSF node-negative ER-positive breast cancer cases with known clinical outcome were used to search for potential genes signifying upregulated NFκB and AP-1 transcriptional activity in association with tamoxifen resistance. The association of these genes with patient outcome was further evaluated using node-negative ER-positive breast cancer cases identified from three other published data sets (Rotterdam, n = 209; Amsterdam, n = 68; Basel, n = 108, each having different patient age and adjuvant tamoxifen treatment characteristics. Results Doses of parthenolide and bortezomib capable of sensitizing the two endocrine resistant breast cancer models to tamoxifen were capable of suppressing NFκB and AP-1 regulated gene expression in combination with tamoxifen and also increased ER recruitment of the transcriptional co-repressor, NCoR. Transcript profiles from the UCSF breast cancer cases revealed three NFκB and AP-1 upregulated genes – cyclin D1, uPA and VEGF – capable of dichotomizing node-negative ER-positive cases into early and late relapsing subsets despite adjuvant tamoxfien therapy and most prognostic for younger age cases. Across the four independent sets of node-negative ER-positive breast cancer cases

  4. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.

    Science.gov (United States)

    Zhou, Hui; Lin-Wang, Kui; Wang, Huiliang; Gu, Chao; Dare, Andrew P; Espley, Richard V; He, Huaping; Allan, Andrew C; Han, Yuepeng

    2015-04-01

    Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors.

  5. Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.

    Science.gov (United States)

    Zhou, Hui; Lin-Wang, Kui; Wang, Huiliang; Gu, Chao; Dare, Andrew P; Espley, Richard V; He, Huaping; Allan, Andrew C; Han, Yuepeng

    2015-04-01

    Anthocyanin pigmentation is an important consumer trait in peach (Prunus persica). In this study, the genetic basis of the blood-flesh trait was investigated using the cultivar Dahongpao, which shows high levels of cyanidin-3-glucoside in the mesocarp. Elevation of anthocyanin levels in the flesh was correlated with the expression of an R2R3 MYB transcription factor, PpMYB10.1. However, PpMYB10.1 did not co-segregate with the blood-flesh trait. The blood-flesh trait was mapped to a 200-kb interval on peach linkage group (LG) 5. Within this interval, a gene encoding a NAC domain transcription factor (TF) was found to be highly up-regulated in blood-fleshed peaches when compared with non-red-fleshed peaches. This NAC TF, designated blood (BL), acts as a heterodimer with PpNAC1 which shows high levels of expression in fruit at late developmental stages. We show that the heterodimer of BL and PpNAC1 can activate the transcription of PpMYB10.1, resulting in anthocyanin pigmentation in tobacco. Furthermore, silencing the BL gene reduces anthocyanin pigmentation in blood-fleshed peaches. The transactivation activity of the BL-PpNAC1 heterodimer is repressed by a SQUAMOSA promoter-binding protein-like TF, PpSPL1. Low levels of PpMYB10.1 expression in fruit at early developmental stages is probably attributable to lower levels of expression of PpNAC1 plus the presence of high levels of repressors such as PpSPL1. We present a mechanism whereby BL is the key gene for the blood-flesh trait in peach via its activation of PpMYB10.1 in maturing fruit. Partner TFs such as basic helix-loop-helix proteins and NAC1 are required, as is the removal of transcriptional repressors. PMID:25688923

  6. MYRF is a membrane-associated transcription factor that autoproteolytically cleaves to directly activate myelin genes.

    Directory of Open Access Journals (Sweden)

    Helena Bujalka

    Full Text Available The myelination of axons is a crucial step during vertebrate central nervous system (CNS development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf, as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.

  7. CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Lau, Wen Min; Doucet, Michele; Huang, David; Weber, Kristy L.; Kominsky, Scott L., E-mail: kominsc@jhmi.edu

    2013-07-26

    Highlights: •The effects of elevated CITED2 on ER function in breast cancer cells are examined. •CITED2 enhances cell growth in the absence of estrogen and presence of tamoxifen. •CITED2 functions as a transcriptional co-activator of ER in breast cancer cells. -- Abstract: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator

  8. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Lushuai [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yanyan; Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Han, Xiao [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Niu, Yuanjie [Chawnshang Chang Sex Hormone Research Center, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300070 (China); Ren, Shancheng, E-mail: renshancheng@gmail.com [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  9. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.

    Science.gov (United States)

    Lin, Jiandie; Wu, Hai; Tarr, Paul T; Zhang, Chen-Yu; Wu, Zhidan; Boss, Olivier; Michael, Laura F; Puigserver, Pere; Isotani, Eiji; Olson, Eric N; Lowell, Bradford B; Bassel-Duby, Rhonda; Spiegelman, Bruce M

    2002-08-15

    The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination. PMID:12181572

  10. BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity

    International Nuclear Information System (INIS)

    Research highlights: → BTG2 associates with AR, androgen causes an increase of the interaction. → BTG2 as a co-repressor inhibits the AR-mediated transcription activity. → BTG2 inhibits the transcription activity and expression of PSA. → An intact 92LxxLL96 motif is essential and necessary for these activities of BTG2, while the 20LxxLL24 motif is not required. → Ectopic expression of BTG2 reduces proliferation of prostate cancer cells. -- Abstract: The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs (20LxxLL24 and 92LxxLL96), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant 20LxxLL24 motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant 92LxxLL96 motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact 92LxxLL96 motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

  11. The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, F.; Petrovicova, I.;

    2008-01-01

    The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition...

  12. Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression.

    Directory of Open Access Journals (Sweden)

    Shadi Jafari

    Full Text Available The mechanism that specifies olfactory sensory neurons to express only one odorant receptor (OR from a large repertoire is critical for odor discrimination but poorly understood. Here, we describe the first comprehensive analysis of OR expression regulation in Drosophila. A systematic, RNAi-mediated knock down of most of the predicted transcription factors identified an essential function of acj6, E93, Fer1, onecut, sim, xbp1, and zf30c in the regulation of more than 30 ORs. These regulatory factors are differentially expressed in antennal sensory neuron classes and specifically required for the adult expression of ORs. A systematic analysis reveals not only that combinations of these seven factors are necessary for receptor gene expression but also a prominent role for transcriptional repression in preventing ectopic receptor expression. Such regulation is supported by bioinformatics and OR promoter analyses, which uncovered a common promoter structure with distal repressive and proximal activating regions. Thus, our data provide insight into how combinatorial activation and repression can allow a small number of transcription factors to specify a large repertoire of neuron classes in the olfactory system.

  13. Transcriptional activation of melanocortin 2 receptor accessory protein by PPARγ in adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Nam Soo; Kim, Yoon-Jin [Department of Biology, Research Institute for Basic Science, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Cho, Si Young [R and D Center, Amore Pacific Corporation, Yongin-si, Gyeonggi-do 446-729 (Korea, Republic of); Lee, Tae Ryong, E-mail: trlee@amorepacific.com [R and D Center, Amore Pacific Corporation, Yongin-si, Gyeonggi-do 446-729 (Korea, Republic of); Kim, Sang Hoon, E-mail: shkim@khu.ac.kr [Department of Biology, Research Institute for Basic Science, Kyung Hee University, Seoul 130-701 (Korea, Republic of)

    2013-09-27

    Highlights: •MRAP enhanced HSL expression. •ACTH-mediated MRAP reduced glycerol release. •PPARγ induced MRAP expression. •PPARγ bound to the MRAP promoter. -- Abstract: Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes.

  14. Transcriptional activation of melanocortin 2 receptor accessory protein by PPARγ in adipocytes

    International Nuclear Information System (INIS)

    Highlights: •MRAP enhanced HSL expression. •ACTH-mediated MRAP reduced glycerol release. •PPARγ induced MRAP expression. •PPARγ bound to the MRAP promoter. -- Abstract: Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes

  15. Bromodomain protein 4 mediates the papillomavirus E2 transcriptional activation function.

    Science.gov (United States)

    Schweiger, Michal-Ruth; You, Jianxin; Howley, Peter M

    2006-05-01

    The papillomavirus E2 regulatory protein has essential roles in viral transcription and the initiation of viral DNA replication as well as for viral genome maintenance. Brd4 has recently been identified as a major E2-interacting protein and, in the case of the bovine papillomavirus type 1, serves to tether E2 and the viral genomes to mitotic chromosomes in dividing cells, thus ensuring viral genome maintenance. We have explored the possibility that Brd4 is involved in other E2 functions. By analyzing the binding of Brd4 to a series of alanine-scanning substitution mutants of the human papillomavirus type 16 E2 N-terminal transactivation domain, we found that amino acids required for Brd4 binding were also required for transcriptional activation but not for viral DNA replication. Functional studies of cells expressing either the C-terminal domain of Brd4 that can bind E2 and compete its binding to Brd4 or short interfering RNA to knock down Brd4 protein levels revealed a role for Brd4 in the transcriptional activation function of E2 but not for its viral DNA replication function. Therefore, these studies establish a broader role for Brd4 in the papillomavirus life cycle than as the chromosome tether for E2 during mitosis. PMID:16611886

  16. ELK3 suppresses angiogenesis by inhibiting the transcriptional activity of ETS-1 on MT1-MMP.

    Science.gov (United States)

    Heo, Sun-Hee; Cho, Je-Yoel

    2014-01-01

    Ets transcription factors play important roles in vasculogenesis and angiogenesis. Knockout of the Ets gene family members in mice resulted in disrupted angiogenesis and malformed vascular systems. In this study, the role and mechanism of ELK3, an Ets factor, in angiogenesis was investigated using ELK3-specific siRNA in human vascular endothelial cells (HUVECs) and in vivo implantation assay. The suppression of ELK3 expression resulted in the reinforcement of VEGF-induced tube formation in HUVECs. The in vivo Matrigel plug assay also showed that ELK3 knockdown resulted in increased angiogenesis. Luciferase activity of the MT1-MMP promoter induced by ETS-1 factor was attenuated ELK3 co-transfection. CHIP assay showed the binding of ELK3 on the MT1-MMP promoter. MT1-MMP knockdown in the ELK3 knockdowned cells resulted in the decrease of tube formation suggesting that MT1-MMP transcriptional repression is required for ELK3-mediated anti-angiogenesis effect. Our data also showed that the suppressive effect of ELK3 on the angiogenesis was partly due to the inhibitory effect of ELK3 to the ETS-1 transcriptional activity on the MT1-MMP promoter rather than direct suppression of ELK3 on the target gene, since the expression level of co-repressor Sin3A is low in endothelial cells. Our results suggest that ELK3 plays a negative role of VEGF-induced angiogenesis through indirectly inhibiting ETS-1 function. PMID:24719561

  17. Mercury Detoxification by Bacteria: Simulations of Transcription Activation and Mercury-Carbon Bond Cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Hao-Bo [ORNL; Parks, Jerry M [ORNL; Johs, Alexander [ORNL; Smith, Jeremy C [ORNL

    2011-01-01

    In this chapter, we summarize recent work from our laboratory and provide new perspective on two important aspects of bacterial mercury resistance: the molecular mechanism of transcriptional regulation by MerR, and the enzymatic cleavage of the Hg-C bond in methylmercury by the organomercurial lyase, MerB. Molecular dynamics (MD) simulations of MerR reveal an opening-and-closing dynamics, which may be involved in initiating transcription of mercury resistance genes upon Hg(II) binding. Density functional theory (DFT) calculations on an active-site model of the enzyme reveal how MerB catalyzes the Hg-C bond cleavage using cysteine coordination and acid-base chemistry. These studies provide insight into the detailed mechanisms of microbial gene regulation and defense against mercury toxicity.

  18. Calmodulin-binding transcription activator (CAMTA) 3 mediates biotic defense responses in Arabidopsis.

    Science.gov (United States)

    Galon, Yael; Nave, Roy; Boyce, Joy M; Nachmias, Dikla; Knight, Marc R; Fromm, Hillel

    2008-03-19

    Calmodulin-binding transcription activator (CAMTA) 3 (also called SR1) is a calmodulin-binding transcription factor in Arabidopsis. Two homozygous T-DNA insertion mutants (camta3-1, camta3-2) showed enhanced spontaneous lesions. Transcriptome analysis of both mutants revealed 6 genes with attenuated expression and 99 genes with elevated expression. Of the latter, 32 genes are related to defense against pathogens (e.g. WRKY33, PR1 and chitinase). Propagation of a virulent strain of the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea were attenuated in both mutants. Moreover, both mutants accumulated high levels of H2O2. We suggest that CAMTA3 regulates the expression of a set of genes involved in biotic defense responses.

  19. Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae.

    Science.gov (United States)

    Manzoor, Irfan; Shafeeq, Sulman; Afzal, Muhammad; Kuipers, Oscar P

    2015-01-01

    In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5'-ATTTGAACATTATTCAAGT-3') in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.

  20. Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Neff, Michael M.

    2011-06-23

    This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

  1. FOXR2 Interacts with MYC to Promote Its Transcriptional Activities and Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Xu Li

    2016-07-01

    Full Text Available By combining the results of a large-scale proteomic analysis of the human transcription factor interaction network with knowledge databases, we identified FOXR2 as one of the top-ranked candidate proto-oncogenes. Here, we show that FOXR2 forms a stable complex with MYC and MAX and subsequently regulates cell proliferation by promoting MYC’s transcriptional activities. We demonstrate that FOXR2 is highly expressed in several breast, lung, and liver cancer cell lines and related patient tumor samples, while reduction of FOXR2 expression in a xenograft model inhibits tumor growth. These results indicate that FOXR2 acts with MYC to promote cancer cell proliferation, which is a potential tumor-specific target for therapeutic intervention against MYC-driven cancers.

  2. RelB, a new Rel family transcription activator that can interact with p50-NF-kappa B.

    OpenAIRE

    Ryseck, R P; Bull, P.; Takamiya, M.; Bours, V; Siebenlist, U; Dobrzanski, P; Bravo, R.

    1992-01-01

    We have identified a serum-inducible gene, relB, which encodes a protein of 558 amino acids containing a region with high similarity to c-Rel and other members of the Rel family. Transcriptional activation analysis of GAL4-RelB fusion proteins in yeast cells reveals that RelB contains in its C-terminal 180 amino acids a transcriptional activation domain. The N-terminal part including the region of similarity with the Rel family shows no detectable transcriptional activity. RelB does not bind ...

  3. In Vitro Anticancer Activity of Phlorofucofuroeckol A via Upregulation of Activating Transcription Factor 3 against Human Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Hyun Ji Eo

    2016-03-01

    Full Text Available Phlorofucofuroeckol A (PFF-A, one of the phlorotannins found in brown algae, has been reported to exert anti-cancer property. However, the molecular mechanism for the anti-cancer effect of PFF-A has not been known. Activating transcription factor 3 (ATF3 has been reported to be associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which PFF-A stimulates ATF3 expression and apoptosis in human colorectal cancer cells. PFF-A decreased cell viability through apoptosis of human colorectal cancer cells. PFF-A increased ATF3 expression through regulating transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by PFF-A was cAMP response element binding protein (CREB, located between positions −147 and −85 of the ATF3 promoter. Inhibition of p38, c-Jun N-terminal kinases (JNK, glycogen synthase kinase (GSK 3β, and IκB kinase (IKK-α blocked PFF-A-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of poly (ADP-ribose polymerase (PARP by PFF-A, while ATF3 overexpression increased PFF-A-mediated cleaved PARP. These results suggest that PFF-A may exert anti-cancer property through inducing apoptosis via the ATF3-mediated pathway in human colorectal cancer cells.

  4. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

    Energy Technology Data Exchange (ETDEWEB)

    Matt Vaughn Greg Harrington Daniel R Bush

    2002-08-06

    This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose

  5. Genetic variants in ABCA1 promoter affect transcription activity and plasma HDL level in pigs.

    Science.gov (United States)

    Dang, Xiao-yong; Chu, Wei-wei; Shi, Heng-chuan; Yu, Shi-gang; Han, Hai-yin; Gu, Shu-Hua; Chen, Jie

    2015-01-25

    Excess accumulation of cholesterol in plasma may result in coronary artery disease. Numerous studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for plasma high density lipoprotein (HDL) formation. Higher plasma levels of HDL are associated with lower risk for cardiovascular disease. Studies of human disease and animal models had shown that an increased hepatic ABCA1 activity relates to an enhanced plasma HDL level. In this study, we hypothesized that functional mutations in the ABCA1 promoter in pigs may affect gene transcription activity, and consequently the HDL level in plasma. The promoter region of ABCA1 was comparatively scanned by direct sequencing with pool DNA of high- and low-HDL groups (n=30 for each group). Two polymorphisms, c. - 608A>G and c. - 418T>A, were revealed with reverse allele distribution in the two groups. The two polymorphisms were completely linked and formed only G-A or A-T haplotypes when genotyped in a larger population (n=526). Furthermore, we found that the G-A/G-A genotype was associated with higher HDL and ABCA1 mRNA level than A-T/A-T genotype. Luciferase assay also revealed that G-A haplotype promoter had higher activity than A-T haplotype. Single-nucleotide mutant assay showed that c.-418T>A was the causal mutation for ABCA1 transcription activity alteration. Conclusively, we identified two completely linked SNPs in porcine ABCA1 promoter region which have influence on the plasma HDL level by altering ABCA1 gene transcriptional activity.

  6. MAPK specificity in the yeast pheromone response independent of transcriptional activation.

    Science.gov (United States)

    Breitkreutz, A; Boucher, L; Tyers, M

    2001-08-21

    The mechanisms whereby different external cues stimulate the same mitogen-activated protein kinase (MAPK) cascade, yet trigger an appropriately distinct biological response, epitomize the conundrum of specificity in cell signaling. In yeast, shared upstream components of the mating pheromone and filamentous growth pathways activate two related MAPKs, Fus3 and Kss1, which in turn regulate programs of gene expression via the transcription factor Ste12. As fus3, but not kss1, strains are impaired for mating, Fus3 exhibits specificity for the pheromone response. To account for this specificity, it has been suggested that Fus3 physically occludes Kss1 from pheromone-activated signaling complexes, which are formed on the scaffold protein Ste5. However, we find that genome-wide expression profiles of pheromone-treated wild-type, fus3, and kss1 deletion strains are highly correlated for all induced genes and, further, that two catalytically inactive versions of Fus3 fail to abrogate the pheromone-induced transcriptional response. Consistently, Fus3 and Kss1 kinase activity is induced to an equivalent extent in pheromone-treated cells. In contrast, both in vivo and in an in vitro-reconstituted MAPK system, Fus3, but not Kss1, exhibits strong substrate selectivity toward Far1, a bifunctional protein required for polarization and G(1) arrest. This effect accounts for the failure to repress G(1)-S specific transcription in fus3 strains and, in part, explains the mating defect of such strains. MAPK specificity in the pheromone response evidently occurs primarily at the substrate level, as opposed to specific kinase activation by dedicated signaling complexes. PMID:11525741

  7. Tyrosine phosphorylation and protein degradation control the transcriptional activity of WRKY involved in benzylisoquinoline alkaloid biosynthesis

    Science.gov (United States)

    Yamada, Yasuyuki; Sato, Fumihiko

    2016-01-01

    Benzylisoquinoline alkaloids (BIQ) are among the most structurally diverse and pharmaceutically valuable secondary metabolites. A plant-specific WRKY-type transcription factor, CjWRKY1, was isolated from Coptis japonica and identified as a transcriptional activator of BIQ biosynthesis. However, the expression of CjWRKY1 gene alone was not sufficient for the activation of genes encoding biosynthetic enzymes. Here, we report the importance of post-translational regulation of CjWRKY1 in BIQ biosynthesis. First, we detected the differential accumulation of CjWRKY1 protein in two cell lines with similar CjWRKY1 gene expression but different levels of accumulated alkaloids. Further investigation of the WRKY protein identified the phosphorylation of the WRKYGQK core domain at Y115. The CjWRKYY115E phosphorylation-mimic mutant showed loss of nuclear localization, DNA-binding activity, and transactivation activity compared to wild-type CjWRKY1. Rapid degradation of the CjWRKY1 protein was also confirmed following treatment with inhibitors of the 26S proteasome and protease inhibitors. The existence of two independent degradation pathways as well as protein phosphorylation suggests the fine-tuning of CjWRKY1 activities is involved in the regulation of biosynthesis of BIQs. PMID:27552928

  8. A trihelix DNA binding protein counterbalances hypoxia-responsive transcriptional activation in Arabidopsis.

    Science.gov (United States)

    Giuntoli, Beatrice; Lee, Seung Cho; Licausi, Francesco; Kosmacz, Monika; Oosumi, Teruko; van Dongen, Joost T; Bailey-Serres, Julia; Perata, Pierdomenico

    2014-09-01

    Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen

  9. A trihelix DNA binding protein counterbalances hypoxia-responsive transcriptional activation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Beatrice Giuntoli

    2014-09-01

    Full Text Available Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1, as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop

  10. Nuclear factor I revealed as family of promoter binding transcription activators

    Directory of Open Access Journals (Sweden)

    Plasari Genta

    2011-04-01

    Full Text Available Abstract Background Multiplex experimental assays coupled to computational predictions are being increasingly employed for the simultaneous analysis of many specimens at the genome scale, which quickly generates very large amounts of data. However, inferring valuable biological information from the comparisons of very large genomic datasets still represents an enormous challenge. Results As a study model, we chose the NFI/CTF family of mammalian transcription factors and we compared the results obtained from a genome-wide study of its binding sites with chromatin structure assays, gene expression microarray data, and in silico binding site predictions. We found that NFI/CTF family members preferentially bind their DNA target sites when they are located around transcription start sites when compared to control datasets generated from the random subsampling of the complete set of NFI binding sites. NFI proteins preferably associate with the upstream regions of genes that are highly expressed and that are enriched in active chromatin modifications such as H3K4me3 and H3K36me3. We postulate that this is a causal association and that NFI proteins mainly act as activators of transcription. This was documented for one member of the family (NFI-C, which revealed as a more potent gene activator than repressor in global gene expression analysis. Interestingly, we also discovered the association of NFI with the tri-methylation of lysine 9 of histone H3, a chromatin marker previously associated with the protection against silencing of telomeric genes by NFI. Conclusion Taken together, we illustrate approaches that can be taken to analyze large genomic data, and provide evidence that NFI family members may act in conjunction with specific chromatin modifications to activate gene expression.

  11. Activating transcription factor 4 and X box binding protein 1 of Litopenaeus vannamei transcriptional regulated white spot syndrome virus genes Wsv023 and Wsv083.

    Directory of Open Access Journals (Sweden)

    Xiao-Yun Li

    Full Text Available In response to endoplasmic reticulum (ER stress, the signaling pathway termed unfolded protein response (UPR is activated. To investigate the role of UPR in Litopenaeus vannamei immunity, the activating transcription factor 4 (designated as LvATF4 which belonged to a branch of the UPR, the [protein kinase RNA (PKR-like ER kinase, (PERK]-[eukaryotic initiation factor 2 subunit alpha (eIF2α] pathway, was identified and characterized. The full-length cDNA of LvATF4 was 1972 bp long, with an open reading frame of 1299 bp long that encoded a 432 amino acid protein. LvATF4 was highly expressed in gills, intestines and stomach. For the white spot syndrome virus (WSSV challenge, LvATF4 was upregulated in the gills after 3 hpi and increased by 1.9-fold (96 hpi compared to the mock-treated group. The LvATF4 knock-down by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Reporter gene assays show that LvATF4 could upregulate the expression of the WSSV gene wsv023 based on the activating transcription factor/cyclic adenosine 3', 5'-monophosphate response element (ATF/CRE. Another transcription factor of L. vannamei, X box binding protein 1 (designated as LvXBP1, has a significant function in [inositol-requiring enzyme-1(IRE1 - (XBP1] pathway. This transcription factor upregulated the expression of the WSSV gene wsv083 based on the UPR element (UPRE. These results suggest that in L. vannamei UPR signaling pathway transcription factors are important for WSSV and might facilitate WSSV infection.

  12. Post-translational Control of the Temporal Dynamics of Transcription Factor Activity Regulates Neurogenesis.

    Science.gov (United States)

    Quan, Xiao-Jiang; Yuan, Liqun; Tiberi, Luca; Claeys, Annelies; De Geest, Natalie; Yan, Jiekun; van der Kant, Rob; Xie, Wei R; Klisch, Tiemo J; Shymkowitz, Joost; Rousseau, Frederic; Bollen, Mathieu; Beullens, Monique; Zoghbi, Huda Y; Vanderhaeghen, Pierre; Hassan, Bassem A

    2016-01-28

    Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. Here, we discover a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, we show that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects. PMID:26824657

  13. Effects of calmodulin on DNA-binding activity of heat shock transcription factor in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

  14. Gas6/Axl pathway promotes tumor invasion through the transcriptional activation of Slug in hepatocellular carcinoma.

    Science.gov (United States)

    Lee, Hsin-Jung; Jeng, Yung-Ming; Chen, Yu-Ling; Chung, Ling; Yuan, Ray-Hwang

    2014-04-01

    Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. Other than the sorafenib treatment, no effective systemic therapy has been available thus far. Most targets in molecularly targeted therapy for cancer are receptor tyrosine kinases (RTKs). Therefore, identifying activated RTKs in HCC is critical for developing new molecularly targeted therapies. Using a phospho-RTK array, we found that Axl is one of the most frequently activated RTKs in liver cancer cell lines. The knockdown of Axl by RNA interference significantly reduced cell migration and invasion in the HCC cell lines HA22T and Mahlavu. Stimulation of HCC cell lines by Axl ligand growth arrest-specific 6 (Gas6) enhanced cell migration and invasion. The Gas6/Axl pathway enhanced the expression of the epithelial-mesenchymal transition-inducing transcription factor Slug, which is essential for the invasion-promoting activity of Axl. Treating HCC cells with the Axl inhibitor bosutinib suppressed Slug expression and decreased the invasiveness of HCC cell lines. These findings indicate that Gas6/Axl regulates tumor invasion through the transcriptional activation of Slug.

  15. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  16. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    Science.gov (United States)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  17. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

    Science.gov (United States)

    Sheikh, Arsheed H; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  18. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

    Science.gov (United States)

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C

    2000-06-01

    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  19. Localizing potentially active post-transcriptional regulations in the Ewing's sarcoma gene regulatory network

    Directory of Open Access Journals (Sweden)

    Delyon Bernard

    2010-11-01

    Full Text Available Abstract Background A wide range of techniques is now available for analyzing regulatory networks. Nonetheless, most of these techniques fail to interpret large-scale transcriptional data at the post-translational level. Results We address the question of using large-scale transcriptomic observation of a system perturbation to analyze a regulatory network which contained several types of interactions - transcriptional and post-translational. Our method consisted of post-processing the outputs of an open-source tool named BioQuali - an automatic constraint-based analysis mimicking biologist's local reasoning on a large scale. The post-processing relied on differences in the behavior of the transcriptional and post-translational levels in the network. As a case study, we analyzed a network representation of the genes and proteins controlled by an oncogene in the context of Ewing's sarcoma. The analysis allowed us to pinpoint active interactions specific to this cancer. We also identified the parts of the network which were incomplete and should be submitted for further investigation. Conclusions The proposed approach is effective for the qualitative analysis of cancer networks. It allows the integrative use of experimental data of various types in order to identify the specific information that should be considered a priority in the initial - and possibly very large - experimental dataset. Iteratively, new dataset can be introduced into the analysis to improve the network representation and make it more specific.

  20. Impact of Heavy Metals on Transcriptional and Physiological Activity of Nitrifying Bacteria.

    Science.gov (United States)

    Kapoor, Vikram; Li, Xuan; Elk, Michael; Chandran, Kartik; Impellitteri, Christopher A; Santo Domingo, Jorge W

    2015-11-17

    Heavy metals can inhibit nitrification, a key process for nitrogen removal in wastewater treatment. The transcriptional responses of amoA, hao, nirK, and norB were measured in conjunction with specific oxygen uptake rate (sOUR) for nitrifying enrichment cultures exposed to different metals (Ni(II), Zn(II), Cd(II), and Pb(II)). There was significant decrease in sOUR with increasing concentrations for Ni(II) (0.03-3 mg/L), Zn(II) (0.1-10 mg/L), and Cd(II) (0.03-1 mg/L) (p amoA and hao decreased when exposed to Ni(II) dosages. Slight up-regulation of amoA, hao, and nirK (0.5-1.5-fold) occurred after exposure to 0.3-3 mg/L Zn(II), although their expression decreased for 10 mg/L Zn(II). With the exception of 1000 mg/L Pb(II), stimulation of all genes occurred on Cd(II) and Pb(II) exposure. While overall the results show that RNA-based function-specific assays can be used as potential surrogates for measuring nitrification activity, the degree of inhibition inferred from sOUR and gene transcription is different. We suggest that variations in transcription of functional genes may supplement sOUR based assays as early warning indicators of upsets in nitrification. PMID:26501957

  1. Phenotypic and transcriptional plasticity directed by a yeast mitogen-activated protein kinase network.

    Science.gov (United States)

    Breitkreutz, Ashton; Boucher, Lorrie; Breitkreutz, Bobby-Joe; Sultan, Mujahid; Jurisica, Igor; Tyers, Mike

    2003-11-01

    The yeast pheromone/filamentous growth MAPK pathway mediates both mating and invasive-growth responses. The interface between this MAPK module and the transcriptional machinery consists of a network of two MAPKs, Fus3 and Kss1; two regulators, Rst1 and Rst2 (a.k.a. Dig1 and Dig2); and two transcription factors, Ste12 and Tec1. Of 16 possible combinations of gene deletions in FUS3, KSS1, RST1, and RST2 in the sigma1278 background, 10 display constitutive invasive growth. Rst1 was the primary negative regulator of invasive growth, while other components either attenuated or enhanced invasive growth, depending on the genetic context. Despite activation of the invasive response by lesions at the same level in the MAPK pathway, transcriptional profiles of different invasive mutant combinations did not exhibit a unified program of gene expression. The distal MAPK regulatory network is thus capable of generating phenotypically similar invasive-growth states (an attractor) from different molecular architectures (trajectories) that can functionally compensate for one another. This systems-level robustness may also account for the observed diversity of signals that trigger invasive growth. PMID:14668360

  2. Viral retasking of hBre1/RNF20 to recruit hPaf1 for transcriptional activation.

    Directory of Open Access Journals (Sweden)

    Gregory J Fonseca

    Full Text Available Upon infection, human adenovirus (HAdV must activate the expression of its early genes to reprogram the cellular environment to support virus replication. This activation is orchestrated in large part by the first HAdV gene expressed during infection, early region 1A (E1A. E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. Previously, we identified hBre1/RNF20 as a target for E1A. The interaction between E1A and hBre1 antagonizes the innate antiviral response by blocking H2B monoubiquitination, a chromatin modification necessary for the interferon (IFN response. Here, we describe a second distinct role for the interaction of E1A with hBre1 in transcriptional activation of HAdV early genes. Furthermore, we show that E1A changes the function of hBre1 from a ubiquitin ligase involved in substrate selection to a scaffold which recruits hPaf1 as a means to stimulate transcription and transcription-coupled histone modifications. By using hBre1 to recruit hPaf1, E1A is able to optimally activate viral early transcription and begin the cycle of viral replication. The ability of E1A to target hBre1 to simultaneously repress cellular IFN dependent transcription while activating viral transcription, represents an elegant example of the incredible economy of action accomplished by a viral regulatory protein through a single protein interaction.

  3. Differential binding activity of the transcription factor LIL-Stat in immature and differentiated normal and leukemic myeloid cells

    NARCIS (Netherlands)

    Tuyt, LML; Bregman, K; Lummen, C; Dokter, WHA; Vellenga, E

    1998-01-01

    Cytokines and growth factors induce activation of the family of signal transducers and activators of transcription (Stats) that directly activate gene expression. Recently, constitutively activated Stat1, Stat3, and Stat5 were identified in nuclear extracts of acute myeloid leukemia (AML) patients,

  4. Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Reavey, Caitlin T; Hickman, Mark J; Dobi, Krista C; Botstein, David; Winston, Fred

    2015-10-01

    Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.

  5. Transcriptional activation of hTERT in breast carcinomas by the Her2-ER81-related pathway.

    Science.gov (United States)

    Vageli, Dimitra; Ioannou, Maria G; Koukoulis, George K

    2009-01-01

    Her2 and ER81 (a member of ETS family) have been suggested to cause a synergistic increase in the transcriptional activation of hTERT. Our study aimed to offer further confirmation in clinical material. We determined the mRNA levels of Her2, ER81, and hTERT, by QRT-PCR, in 43 breast carcinomas. In the specimens showing hTERT transcriptional activation, Her2 and ER81 were increased in statistically significant tumor subgroups (61% and 79% correspondingly). The 86% of specimens with both Her2 and ER81 increased expression showed hTERT transcriptional activation. Synchronous transcriptional activation of hTERT, Her2, and ER81 elevated expression was noted in 42% of the samples. In conclusion, we agree with a previous study that Her2 overexpression may increase the hTERT transcriptional activation. Our data indicate that the mechanism may involve Her2-ER81 interaction(s) and that the activation of hTERT could be mainly mediated by transcriptional activation of ER81.

  6. Conserved and Diverged Functions of the Calcineurin-Activated Prz1 Transcription Factor in Fission Yeast.

    Science.gov (United States)

    Chatfield-Reed, Kate; Vachon, Lianne; Kwon, Eun-Joo Gina; Chua, Gordon

    2016-04-01

    Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeastSchizosaccharomyces pombe Genome-wide studies of theCrz1and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This article identifies an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2and tunicamycin treatment, as well as a∆pmr1genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of theSaccharomyces cerevisiaeorthologCrz1 These genes were functionally enriched forCrz1-conserved processes such as cell-wall biosynthesis. Overexpression ofprz1(+)increased resistance to the cell-wall degradation enzyme zymolyase, likely from upregulation of theO-mannosyltransferase encoding geneomh1(+) Loss ofomh1(+)abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss ofprz1(+)resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the∆prz1strain was abrogated by the loss ofgsf2(+)orcbf12(+) This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell-wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes betweenCrz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional regulatory network. PMID:26896331

  7. Transcriptional activation of the nitrogenase promoter in vitro: adenosine nucleotides are required for inhibition of NIFA activity by NIFL.

    Science.gov (United States)

    Eydmann, T; Söderbäck, E; Jones, T; Hill, S; Austin, S; Dixon, R

    1995-03-01

    The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor sigma 54 (sigma N). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by sigma 54-containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch. PMID:7868590

  8. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

    Science.gov (United States)

    Wang, Fangfang; Okawa, Hiroko; Kamano, Yuya; Niibe, Kunimichi; Kayashima, Hiroki; Osathanon, Thanaphum; Pavasant, Prasit; Saeki, Makio; Yatani, Hirofumi; Egusa, Hiroshi

    2015-01-01

    Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation

  9. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

    Directory of Open Access Journals (Sweden)

    Fangfang Wang

    Full Text Available Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR. Expression vectors that contained the Tet operator and amelogenin-coding (Amelx cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx. MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP, osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional

  10. Essential role of RelA Ser311 phosphorylation by ζPKC in NF-κB transcriptional activation

    OpenAIRE

    Duran, Angeles; Diaz-Meco, María T.; Moscat, Jorge

    2003-01-01

    The activation of the transcription factor NF-κB is central to the control of the cellular response triggered by many stimuli. Once released from the inhibitory molecule IκB, NF-κB is translocated to the nucleus, and it has to be phosphorylated to activate transcription. In ζ protein kinase C (PKC)-deficient cells, NF-κB is transcriptionally inactive and the phosphorylation of the RelA subunit in response to tumor necrosis factor (TNF-α) is severely impaired. In vitro assays showed that ζPKC ...

  11. A dual role for zinc fingers in both DNA binding and zinc sensing by the Zap1 transcriptional activator

    OpenAIRE

    Bird, Amanda J.; Zhao, Hui; Luo, Huan; Jensen, Laran T.; Srinivasan, Chandra; Evans-Galea, Marguerite; Winge, Dennis R.; Eide, David J.

    2000-01-01

    The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change...

  12. Regulation of Transcription through Light-Activation and Light-Deactivation of Triplex-Forming Oligonucleotides in Mammalian Cells

    OpenAIRE

    Govan, Jeane M.; Uprety, Rajendra; Hemphill, James; Lively, Mark O.; Deiters, Alexander

    2012-01-01

    Triplex-forming oligonucleotides (TFOs) are efficient tools to regulate gene expression through the inhibition of transcription. Here, nucleobase-caging technology was applied to the first temporal regulation of transcription through light-activated TFOs. Through site-specific incorporation of caged thymidine nucleotides, the TFO:DNA triplex formation is blocked, rendering the TFO inactive. However, after a brief UV irradiation, the caging groups are removed, activating the TFO, and leading t...

  13. Autogenous regulation of ethanolamine utilization by a transcriptional activator of the eut operon in Salmonella typhimurium.

    OpenAIRE

    Roof, D M; Roth, J R

    1992-01-01

    The genes required for use of ethanolamine as a carbon and nitrogen source are encoded by a single operon (eut) whose expression is induced by the simultaneous presence of both ethanolamine and cobalamin (vitamin B12). The action of B12 as an inducer of this operon reflects the fact that this cofactor is required by the degradative enzyme ethanolamine lyase (eutBC). The eutR gene encodes a protein that activates transcription of the eut operon in response to the simultaneous presence of B12 a...

  14. Sequential Recruitment and Combinatorial Assembling of Multiprotein Complexes in Transcriptional Activation

    Science.gov (United States)

    Lemaire, Vincent; Lee, Chiu Fan; Lei, Jinzhi; Métivier, Raphaël; Glass, Leon

    2006-05-01

    In human cells, estrogenic signals induce cyclical association and dissociation of specific proteins with the DNA in order to activate transcription of estrogen-responsive genes. These oscillations can be modeled by assuming a large number of sequential reactions represented by linear kinetics with random kinetic rates. Application of the model to experimental data predicts robust binding sequences in which proteins associate with the DNA at several different phases of the oscillation. Our methods circumvent the need to derive detailed kinetic graphs, and are applicable to other oscillatory biological processes involving a large number of sequential steps.

  15. Multiple cooperative interactions constrain BPV-1 E2 dependent activation of transcription.

    OpenAIRE

    Sowden, M; Harrison, S; Ashfield, R.; Kingsman, A J; Kingsman, S M

    1989-01-01

    Transcription directed by the BPV-1 long control region (LCR) is conditional upon activation by the virally encoded E2 protein. Within the 1.0 kb LCR there are five separate regions, A to E, that contain E2 responsive enhancers. The smallest functional region, A, is only 38 bp and contains two copies of the consensus sequence ACC(N)6GGT that is known to function as an E2 binding site in vitro. We show that a pair of these constitutes a minimal functional E2 responsive enhancer element but tha...

  16. Efficient activation of transcription in yeast by the BPV1 E2 protein.

    OpenAIRE

    Stanway, C A; Sowden, M P; Wilson, L.E.; Kingsman, A J; Kingsman, S M

    1989-01-01

    The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an...

  17. Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    Energy Technology Data Exchange (ETDEWEB)

    Iwahara, Naotoshi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Hisahara, Shin; Hayashi, Takashi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University, Sapporo 060-8556 (Japan); Horio, Yoshiyuki, E-mail: horio@sapmed.ac.jp [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  18. Separation of the transcriptional activation and replication functions of the bovine papillomavirus-1 E2 protein.

    OpenAIRE

    Winokur, P. L.; McBride, A A

    1992-01-01

    Replication of bovine papillomavirus-1 (BPV-1) DNA requires two viral gene products, the E1 protein and the full-length E2 protein. The 48 kDa E2 protein is a site-specific DNA-binding protein that binds to several sites which lie adjacent to the BPV-1 origin of replication. The 85 amino acid C-terminal domain contains the specific DNA binding and dimerization properties of the protein. The approximately 200 amino acid N-terminal domain is crucial for transcriptional activation. Both of these...

  19. Negative regulation of TLR-signaling pathways by activating transcription factor-3.

    Science.gov (United States)

    Whitmore, Mark M; Iparraguirre, Amaya; Kubelka, Lindsey; Weninger, Wolfgang; Hai, Tsonwin; Williams, Bryan R G

    2007-09-15

    Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.

  20. Germ cell nuclear factor directly represses the transcription of peroxisome proliferator-activated receptor delta gene

    Institute of Scientific and Technical Information of China (English)

    Chengqiang He; Naizheng Ding; Jie Kang

    2008-01-01

    Germ cell nuclear factor (GCNF) is a transcription factor that can repress gene transcription and plays an important role during spermatogenesis. Peroxisome proliferator-activated receptor delta (PPARδ) is a nuclear hormone receptor belonging to the steroid receptor superfamily.It can activate the expression of many genes,including those involved in lipid metabolism.In this report,we showed that GCNF specifically interacts with PPARδ promoter.Overexpression of GCNF in African green monkey SV40 transformed kidney fibroblast COS7 cells and mouse embryo fibroblast NIH 3T3 cells represses the activity of PPARδ promoter.The mutation of GCNF response element in PPARδ promoter relieves the repression in NIH 3T3 cells and mouse testis.Moreover,we showed that GCNF in nuclear extracts of mouse testis is able to bind to PPARδ promoter directly.We also found that GCNF and PPARδ mRNA were expressed with different patterns in mouse testis by in situ hybridization.These results suggested that GCNF might be a negative regulator of PPARδ gene expression through its direct interaction with PPARδ promoter in mouse testis.

  1. Structural Features and Transcriptional Activity of Chicken PPARs (α, β, and γ

    Directory of Open Access Journals (Sweden)

    Ichiro Takada

    2013-01-01

    Full Text Available While an understanding of lipid metabolism in chickens is critical for a further improvement of food production, there are few studies concerning differences in lipid metabolism mechanisms between chickens and other species at a molecular level. Chickens have three PPAR gene subtypes (α, β, and γ that function differently from those present in humans and mice. The chicken PPAR-gamma (cPPARγ gene is shorter than that in humans and lacks a γ2 isoform. Moreover, in serum-free media, cPPARγ shows high transcriptional activity without exogenous ligands. Luciferase reporter assays were used to examine the effect of sera on cPPAR transcriptional activities and showed that adult bovine serum and chicken serum highly activate cPPARα and β functions. Moreover, we found that bezafibrate induces the transactivation function of cPPARβ, but not human PPARδ (human PPARβ ortholog. This ligand selectivity relies on one amino acid residue (chicken: Val419, human: Met444. These results show the possibilities for unique functions of cPPARs on chicken-specific lipid glucose metabolism. As such, a better understanding of the molecular mechanisms of lipid metabolism in chickens could result in higher productivity for the poultry industry.

  2. Dynamic transcription factor activity and networks during ErbB2 breast oncogenesis and targeted therapy.

    Science.gov (United States)

    Weiss, M S; Peñalver Bernabé, B; Shin, S; Asztalos, S; Dubbury, S J; Mui, M D; Bellis, A D; Bluver, D; Tonetti, D A; Saez-Rodriguez, J; Broadbelt, L J; Jeruss, J S; Shea, L D

    2014-12-01

    Tissue development and disease progression are multi-stage processes controlled by an evolving set of key regulatory factors, and identifying these factors necessitates a dynamic analysis spanning relevant time scales. Current omics approaches depend on incomplete biological databases to identify critical cellular processes. Herein, we present TRACER (TRanscriptional Activity CEll aRrays), which was employed to quantify the dynamic activity of numerous transcription factor (TFs) simultaneously in 3D and networks for TRACER (NTRACER), a computational algorithm that allows for cellular rewiring to establish dynamic regulatory networks based on activity of TF reporter constructs. We identified major hubs at various stages of culture associated with normal and abnormal tissue growth (i.e., ELK-1 and E2F1, respectively) and the mechanism of action for a targeted therapeutic, lapatinib, through GATA-1, which were confirmed in human ErbB2 positive breast cancer patients and human ErbB2 positive breast cancer cell lines that were either sensitive or resistant to lapatinib.

  3. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong, E-mail: yelihong@nankai.edu.cn [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2013-05-03

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

  4. PEA3 activates VEGF transcription in T47D and SKBR3 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Dong Hua; Bobin Chen; Mei Bai; Hao Yu; Xiaohong Wu; Wei Jin

    2009-01-01

    Vascular endothelial growth factor(VEGF)is a potent stimulator of angiogenesis and a prognostic factor for many tumors,including those of endocrine-responsive tissues such as the breast and uterus.In this study,we found that overexpression of PEA3 could increase VEGF mRNA levels and VEGF promoter activity in human T47D and SKBR3 breast cancer cells.Chromatin immunoprecipitation assay demonstrated that PEA3 could bind to the VEGF promoter in the cells transfected with PEA3 expression vector.PEA3 small interfering RNA attenuated VEGF promoter activity and the binding of PEA3 to the VEGF promoter in T47D and SKBR3 cells.These results indicated that PEA3 could activate VEGF promoter transcription.

  5. STAT5 transcriptional activity is impaired by LIF in a mammary epithelial cell line.

    Science.gov (United States)

    Granillo, Agustina Rodriguez; Boffi, Juan Carlos; Barañao, Lino; Kordon, Edith; Pecci, Adali; Guberman, Alejandra

    2007-05-11

    Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation. We have also found that STAT5 activity is negatively modulated by this cytokine, both on a gene whose expression is induced, as well as on a promoter repressed by STAT5. Besides, our results show that lactogenic hormones increase LIF effect on gene induction without modifying STAT3 phosphorylation state. Our findings strongly suggest that there is crosstalk between STAT5 and STAT3 pathways that could modulate their ability to regulate gene expression.

  6. Signal transducer and activator of transcription 3 regulation by novel binding partners

    Institute of Scientific and Technical Information of China (English)

    Tadashi; Matsuda; Ryuta; Muromoto; Yuichi; Sekine; Sumihito; Togi; Yuichi; Kitai; Shigeyuki; Kon; Kenji; Oritani

    2015-01-01

    Signal transducers and activators of transcription(STATs) mediate essential signals for various biological processes,including immune responses,hematopoiesis,and neurogenesis. STAT3,for example,is involved in the pathogenesis of various human diseases,including cancers,autoimmune and inflammatory disorders. STAT3 activation is therefore tightly regulated at multiple levels to prevent these pathological conditions. A number of proteins have been reported to associate with STAT3 and regulate its activity. These STAT3-interacting proteins function to modulate STAT3-mediated signaling at various steps and mediate the crosstalk of STAT3 with other cellular signaling pathways. This article reviews the roles of novel STAT3 binding partners such as DAXX,zipperinteracting protein kinase,Krüppel-associated box-associated protein 1,Y14,PDZ and LIM domain 2 and signal transducing adaptor protein-2,in the regulation of STAT3-mediated signaling.

  7. E1A activates transcription of p73 and Noxa to induce apoptosis.

    Science.gov (United States)

    Flinterman, Marcella; Guelen, Lars; Ezzati-Nik, Samira; Killick, Richard; Melino, Gerry; Tominaga, Kazuya; Mymryk, Joe S; Gäken, Joop; Tavassoli, Mahvash

    2005-02-18

    p73, a member of the p53 family of proteins, transcriptionally activates a number of genes involved in the control of cell cycle and apoptosis. Overexpression of p73 was detected in a large number of primary head and neck cancers, and in the established cell lines examined, these all contained inactivating p53 mutations. The significance of p73 overexpression in the pathogenesis of head and neck cancer is currently unclear. We have shown that the expression of adenovirus 5 E1A in a panel of head and neck cancer cell lines induces apoptosis independently of their p53 status. In this study we examined the role of p73 and its transcriptional targets in E1A-mediated induction of apoptosis. E1A expression resulted in significant activation of the TAp73 promoter but had no effect on the alternative, DeltaNp73 promoter. E1A also increased expression of endogenous TAp73 mRNA and protein. E1A mutants lacking the p300- and/or pRB-binding sites showed reduced ability to activate the TAp73 promoter. Additionally, mutations in the E2F1-binding sites in the TAp73 promoter impaired activation by E1A. Importantly, expression of the 13S isoform of E1A substantially induced the p53 apoptotic target Noxa in several p53-deficient cancer cell lines. Our results indicate that E1A activation of p73 and the p53 apoptotic target Noxa can occur in the absence of a functional p53. This activation is likely to play a key role in the mechanism of p53-independent apoptosis induced by E1A in some cancers and may provide an avenue for future cancer therapies. PMID:15572378

  8. A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta.

    Science.gov (United States)

    Zhang, Lei; Davies, Laura J; Elling, Axel A

    2015-01-01

    Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-β-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity.

  9. Menin and RNF20 recruitment is associated with dynamic histone modifications that regulate signal transducer and activator of transcription 1 (STAT1-activated transcription of the interferon regulatory factor 1 gene (IRF1

    Directory of Open Access Journals (Sweden)

    Buro Lauren J

    2010-09-01

    Full Text Available Abstract Background Signal transducer and activator of transcription (STAT activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1. Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFNγ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

  10. Immunolocalization of anti-hsf1 to the acetabular glands of infectious schistosomes suggests a non-transcriptional function for this transcriptional activator.

    Directory of Open Access Journals (Sweden)

    Kenji Ishida

    Full Text Available Schistosomiasis is a chronically debilitating disease caused by parasitic worms of the genus Schistosoma, and it is a global problem affecting over 240 million people. Little is known about the regulatory proteins and mechanisms that control schistosome host invasion, gene expression, and development. Schistosome larvae, cercariae, are transiently free-swimming organisms and infectious to man. Cercariae penetrate human host skin directly using proteases that degrade skin connective tissue. These proteases are secreted from anucleate acetabular glands that contain many proteins, including heat shock proteins. Heat shock transcription factors are strongly conserved activators that play crucial roles in the maintenance of cell homeostasis by transcriptionally regulating heat shock protein expression. In this study, we clone and characterize the schistosome Heat shock factor 1 gene (SmHSF1. We verify its ability to activate transcription using a modified yeast one-hybrid system, and we show that it can bind to the heat shock binding element (HSE consensus DNA sequence. Our quantitative RT-PCR analysis shows that SmHSF1 is expressed throughout several life-cycle stages from sporocyst to adult worm. Interestingly, using immunohistochemistry, a polyclonal antibody raised against an Hsf1-peptide demonstrates a novel localization for this conserved, stress-modulating activator. Our analysis suggests that schistosome Heat shock factor 1 may be localized to the acetabular glands of infective cercariae.

  11. Activation of CXCL-8 Transcription by Hepatitis E Virus ORF-1 via AP-1

    Directory of Open Access Journals (Sweden)

    Zhubing Li

    2015-01-01

    Full Text Available Hepatitis E virus (HEV is a small nonenveloped single-stranded positive-sense RNA virus and is one of the major causes for acute hepatitis worldwide. CXCL-8 is a small multifunctional proinflammatory chemokine. It was reported recently that HEV infection significantly upregulates CXCL-8 gene expression. In this study, we investigated the mechanism of HEV-induced CXCL-8 transcriptional activation. Using CXCL-8 promoter reporters of different lengths ranging from −1400 to −173, we showed that −173 promoter has the highest promoter activity in the presence of HEV genomic RNA, indicating that the −173 promoter contains sequences responsible for CXCL-8 activation by HEV. Ectopic expression of the ORF-1 protein can upregulate the −173 CXCL-8 promoter activity. In contrast, expression of the ORF-2 protein suppresses the CXCL-8 promoter activity and expression of the ORF-3 protein has no effect on the CXCL-8 promoter activity. We further showed that AP-1 is required for CXCL-8 activation because neither HEV genomic RNA nor the ORF-1 protein can upregulate the −173 CXCL-8 promoter in the absence of the AP-1 binding sequence. Taken together, our results showed that HEV and HEV ORF-1 protein activate the CXCL-8 promoter via AP-1. This novel function of HEV ORF-1 protein should contribute to our understanding of HEV-host interactions and HEV-associated pathogenesis.

  12. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding

    International Nuclear Information System (INIS)

    Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter

  13. Genome-wide analysis of basic leucine zipper transcription factor families in Arabidopsis thaliana, Oryza saliva and Populus trichocarpa

    Institute of Scientific and Technical Information of China (English)

    JI Qian; ZHANG Liang-sheng; WANG Yi-fei; WANG Jian

    2009-01-01

    The basic leucine zipper (bZIP) transcription factors form a large gene family that is important in pathogen defense, light and stress signaling, etc. The Completed whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana), rice (Oryza saliva) and poplar (Populus trichocarpa) constitute a valuable resource for genome-wide analysis and genomic comparative analysis, as they are representatives of the two major evolutionary lineages within the angiosperms: the monocotyledons and the dicotyledons. In this study, bioinformatics analysis identified 74, 89 and 88 bZIP genes respectively in Arabidopsis, rice and poplar. Moreover, a comprehensive overview of this gene family is presented, including the gene structure, phylogeny, chromosome distribution, conserved motifs. As a result, the plant bZIPs were organized into 10 subfamilies on basis of phylogenetic relationship. Gene duplication events during the family evolution history were also investigated. And it was further concluded that chromosomal/segmental duplication might have played a key role in gene expansion of bZIP gene family.

  14. Transcriptional activation of glutathione pathways and role of glucose homeostasis during copper imbalance.

    Science.gov (United States)

    Quiroz, Natalia; Rivas, Nicole; del Pozo, Talía; Burkhead, Jason; Suazo, Miriam; González, Mauricio; Latorre, Mauricio

    2015-04-01

    Copper is an essential micronutrient for organism health. Dietary changes or pathologies linked to this metal induce changes in intracellular glutathione concentrations. Here, we studied the transcriptional activation of glutathione pathways in Jurkat cell lines, analyzing the effect of change in glucose homeostasis during a physiological and supra-physiological copper exposure. An immortalized line of human T lymphocyte cell line (Jurkat) was exposed to different copper and glucose conditions to mimic concentrations present in human blood. We applied treatments for 6 (acute) and 24 h (sustained) to 2 µM (physiological) or 20 µM (supra-physiological, Wilson disease scenario) of CuSO4 in combination with 25 mg/dL (hypoglycemia), 100 mg/dL (normal) and 200 mg/dL (hyperglycemia, diabetes scenario) of glucose. The results indicate that a physiological concentration of copper exposure does not induce transcriptional changes in the glutathione synthesis pathway after 6 or 24 h. The G6PDH gene (regeneration pathway), however, is induced during a supra-physiological copper condition. This data was correlated with the viability assays, where fluctuation in both glucose conditions (hypo and hyperglycemia scenario) affected Jurkat proliferation when 20 µM of CuSO4 was added to the culture media. Under a copper overload condition, the transcription of a component of glutathione regeneration pathway (G6PDH gene) is activated in cells chronically exposed to a hyperglycemia scenario, indicating that fluctuations in glucose concentration impact the resistance against the metal. Our findings illustrate the importance of glucose homeostasis during copper excess.

  15. Pronounced cohabitation of active immunoglobulin genes from three different chromosomes in transcription factories during maximal antibody synthesis.

    Science.gov (United States)

    Park, Sung-Kyun; Xiang, Yougui; Feng, Xin; Garrard, William T

    2014-06-01

    To understand the relationships between nuclear organization and gene expression in a model system, we employed three-dimensional imaging and chromatin immunoprecipitation (ChIP)-chromosome conformation capture (3C) techniques to investigate the topographies of the immunoglobulin (Ig) genes and transcripts during B-cell development. Remarkably, in plasma cells, when antibody synthesis peaks, active Ig genes residing on three different chromosomes exhibit pronounced colocalizations in transcription factories, often near the nuclear periphery, and display trans-chromosomal enhancer interactions, and their transcripts frequently share interchromatin trafficking channels. Conceptually, these features of nuclear organization maximize coordinated transcriptional and transcript trafficking control for potentiating the optimal cytoplasmic assembly of the resulting translation products into protein multimers.

  16. Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

    OpenAIRE

    McCulloch, Vicki; Shadel, Gerald S.

    2003-01-01

    A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate tha...

  17. BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Xu-Dong [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Meng, Qing-Hui [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Xu, Jia-Ying; Jiao, Yang [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Ge, Chun-Min [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Jacob, Asha; Wang, Ping [North Shore University Hospital-Long Island Jewish Medical Center and The Feinstein Institute for Medical Research, Manhasset, NY 11030 (United States); Rosen, Eliot M [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Fan, Saijun, E-mail: sjfan@suda.edu.cn [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China)

    2011-01-28

    Research highlights: {yields} BTG2 associates with AR, androgen causes an increase of the interaction. {yields} BTG2 as a co-repressor inhibits the AR-mediated transcription activity. {yields} BTG2 inhibits the transcription activity and expression of PSA. {yields} An intact {sup 92}LxxLL{sup 96} motif is essential and necessary for these activities of BTG2, while the {sup 20}LxxLL{sup 24} motif is not required. {yields} Ectopic expression of BTG2 reduces proliferation of prostate cancer cells. -- Abstract: The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ({sup 20}LxxLL{sup 24} and {sup 92}LxxLL{sup 96}), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5{alpha}-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant {sup 20}LxxLL{sup 24} motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant {sup 92}LxxLL{sup 96} motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact {sup 92}LxxLL{sup 96} motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

  18. Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions.

    Science.gov (United States)

    Sakai, H; Yasugi, T; Benson, J D; Dowhanick, J J; Howley, P M

    1996-03-01

    The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor. PMID:8627680

  19. GFP tracking transcriptional activity endogenous p53: vector construction and application in genotoxicity detection

    Institute of Scientific and Technical Information of China (English)

    ZENG Wei-sen; LUO Chen; XIE Wei-bing; CHEN Han-yuan

    2001-01-01

    To establish a sensitive.and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H202 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H202, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP, hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.

  20. Transcription Activity of Ectogenic Human Carcinoembryonic Antigen Promoter in Lung Adenocarcinoma Cells A549

    Institute of Scientific and Technical Information of China (English)

    XIONG Weining; FANG Huijuan; XU Yongjian; XIONG Shendao; CAO Yong; SONG Qingfeng; ZENG Daxiong; ZHANG Huilan

    2006-01-01

    The transcription activity of ectogenic human carcinoembryonic antigen (CEA) promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein (GFP) driven by CEA promoter and human cytomegalovirus (CMV) promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed (4.08±0.63) % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [(43.27±3.54) %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.

  1. SUMOylation modulates the transcriptional activity of androgen receptor in a target gene and pathway selective manner.

    Science.gov (United States)

    Sutinen, Päivi; Malinen, Marjo; Heikkinen, Sami; Palvimo, Jorma J

    2014-07-01

    Androgen receptor (AR) plays an important regulatory role in prostate cancer. AR's transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates AR's interaction with the chromatin and the receptor's target gene selection.

  2. Conserved hypothetical BB0462 protein enhances the transcription activity of oppAV promoter

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Borrelia burgdorferi BB0462 ORF encodes an unknown functional protein with 110 amino acids.A BLAST search in protein databases and the secondary structure being predicted by the program JUFO showed that the conserved hypothetical BB0462 protein was similar to the members of the YbaB protein family in both amino acid composition and protein structure.The co-transformation of BB0462 ORF and oppA upstream regulation DNA into E.coli host cells and β-galactosidase activity assay demonstrated that the BB0462 protein enhanced the transcriptional activity of the oppAV promoter,but does not affect those of oppAⅠ,Ⅱ,Ⅲ and Ⅳ promoters.Analysis of DNA retardation and competitive repression also confirmed that the BB0462 protein bound to the 409 bp upstream regulation DNA fragment close to the initiation codon of the oppAV gene.All data in our study suggested that the BB0462 protein was involved in the transcriptional regulation of the oppAV gene

  3. ACETYLATION INCREASES EWS-FLI1 DNA BINDING AND TRANSCRIPTIONAL ACTIVITY

    Directory of Open Access Journals (Sweden)

    Silke eSchlottmann

    2012-09-01

    Full Text Available Ewing Sarcoma (ES is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant posttranslational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors, and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with histone deacetylase inhibitors (HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in COS7 cells. However, our data that evaluates the acetylation of ful-length EWS-FLI1 remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  4. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers.

    Science.gov (United States)

    Bultmann, Sebastian; Morbitzer, Robert; Schmidt, Christine S; Thanisch, Katharina; Spada, Fabio; Elsaesser, Janett; Lahaye, Thomas; Leonhardt, Heinrich

    2012-07-01

    Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2'-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes.

  5. Identification and Characterization of Reverse Transcriptase Domain of Transcriptionally Active Retrotransposons in Wheat Genomes

    Institute of Scientific and Technical Information of China (English)

    Yi-Miao TANG; You-Zhi MA; Lian-Cheng LI; Xing-Guo YE

    2005-01-01

    To clarify activation characterization of wheat (Triticum aestivum L.) retrotransposons, transcriptionally active Ty1-copia retrotransposons were found in wheat by using RT-PCR to amplify the RT domain. Sequence analysis of random RT-PCR clones reveals that Ty1-copia retrotransposons are highly heterogeneous and can be divided into at least four groups, which are tentatively named TaRT-1 to TaRT-4.Dot blot hybridization indicates that TaRT- 1 exists in the wheat genome as multiple copies (at 30 000 copies/a hexaploid genome (ABD)). Northern blot hybridization showed that TaRT-1 is only expressed at a low level under normal conditions in seedlings, but at a high level when induced by powdery mildew fungus, jasmonic acid (JA) and salicylic acid (SA). These results suggest that the TaRT-1 expression is highly sensitive to biotic and abiotic stresses.

  6. Hes-1, a known transcriptional repressor, acts as a transcriptional activator for the human acid alpha-glucosidase gene in human fibroblast cells.

    Science.gov (United States)

    Yan, Bo; Raben, Nina; Plotz, Paul H

    2002-03-01

    Hes-1, the mammalian homologue 1 of Drosophila hairy and Enhancer of split proteins, belongs to a family of basic helix-loop-helix proteins that are essential to neurogenesis, myogenesis, hematopoiesis, and sex determination. Hes-1 is a transcriptional repressor for a number of known genes including the human acid alpha-glucosidase (GAA) gene as we have previously shown in Hep G2 cells. The human GAA gene encodes the enzyme for glycogen breakdown in lysosomes, deficiency of which results in Glycogen Storage Disease type II (Pompe syndrome). Using constructs containing the DNA element that demonstrates repressive activity in Hep G2 cells and conditions in which the same transcription factors, Hes-1 and YY1, bind, we have shown that this element functions as an enhancer in human fibroblasts. Site-directed mutagenesis and overexpression of Hes-1 showed that Hes-1 functions as a transcriptional activator. The dual function of Hes-1 we have found is likely to contribute to the subtle tissue-specific control of this housekeeping gene.

  7. Glucocorticoid Receptor Transcriptional Activation via the BRG1-Dependent Recruitment of TOP2β and Ku70/86.

    Science.gov (United States)

    Trotter, Kevin W; King, Heather A; Archer, Trevor K

    2015-08-01

    BRG1, the central ATPase of the human SWI/SNF complex, is critical for biological functions, including nuclear receptor (NR)-regulated transcription. Analysis of BRG1 mutants demonstrated that functional motifs outside the ATPase domain are important for transcriptional activity. In the course of experiments examining protein interactions mediated through these domains, Ku70 (XRCC6) was found to associate with a BRG1 fragment encompassing the conserved helicase-SANT-associated (HSA) and BRK domains of BRG1. Subsequent transcriptional activation assays and chromatin immunoprecipitation studies showed that Ku70/86 and components of the topoisomerase IIβ (TOP2β)/poly(ADP ribose) polymerase 1 (PARP1) complex are necessary for NR-mediated SWI/SNF-dependent transcriptional activation from endogenous promoters. In addition to establishing Ku-BRG1 binding and TOP2β/PARP1 recruitment by nuclear receptor transactivation, we demonstrate that the transient appearance of glucocorticoid receptor (GR)/BRG1-dependent, TOP2β-mediated double-strand DNA breaks is required for efficient GR-stimulated transcription. Taken together, these results suggest that a direct interaction between Ku70/86 and BRG1 brings together SWI/SNF remodeling capabilities and TOP2β activity to enhance the transcriptional response to hormone stimulation.

  8. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    Science.gov (United States)

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

  9. Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics.

    Directory of Open Access Journals (Sweden)

    Katie Porter

    Full Text Available The primary role of Actin-Depolymerizing Factors (ADFs is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1. These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.

  10. Intermedin/adrenomedullin 2 is a stress-inducible gene controlled by activating transcription factor 4.

    Science.gov (United States)

    Kovaleva, Irina E; Garaeva, Alisa A; Chumakov, Peter M; Evstafieva, Alexandra G

    2016-09-15

    Intermedin or adrenomedullin 2 is a set of calcitonin-related peptides with a putative tumor angiogenesis promoting activity that are formed by proteolytic processing of the ADM2 gene product. It has been proposed that the ADM2 gene is regulated by the estrogen response element (ERE) and hypoxia response elements (HRE) found within its promoter region. In the present study we reveal a functional mechanism by which ADM2 participates in the unfolded protein response (UPR) and in responses to the mitochondrial respiration chain inhibition. We show that the ADM2 gene is controlled by activating transcription factor 4 (ATF4), the principal regulator of the integrated stress response (ISR). The upregulation of ADM2 mRNA could be prevented by the pharmacological ISR inhibitor ISRIB and by the downregulation of ATF4 with specific shRNA, while ectopic expression of ATF4 cDNA resulted in a notable increase in ADM2 gene transcription. A potential ATF4-binding site was identified in the coding region of the ADM2 gene and the requirement of this site during the ATF4-mediated ADM2 gene promoter activation was validated by the luciferase reporter assay. Mutagenesis of the putative ATF4-response element prevented the induction of luciferase activity in response to ATF4 overproduction, as well as in response to mitochondrial electron transfer chain inhibition by piericidin A and ER stress induction by tunicamycin and brefeldin A. Since ADM2 was shown to inhibit ATF4 expression during myocardial ER stress, a feedback mechanism could be proposed for the ADM2 regulation under ER stress conditions. PMID:27328454

  11. Transcriptional Activation by Wild-Type But Not Transforming Mutants of the p53 Anti-Oncogene

    OpenAIRE

    Raycroft, Loretta; Wu, Hongyun; Lozano, Guillermina

    1990-01-01

    The protein encoded by the wild-type p53 proto-oncogene has been shown to suppress transformation, whereas certain mutations that alter p53 become transformation competent. Fusion proteins between p53 and the GAL4 DNA binding domain were made to anchor p53 to a DNA target sequence and to allow measurement of transcriptional activation of a reporter plasmid. The wild-type p53 stimulated transcription in this assay, but two transforming mutations in p53 were unable to act as transcriptional act...

  12. Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.

    Science.gov (United States)

    O'Shea, Charlotte; Kryger, Mikael; Stender, Emil G P; Kragelund, Birthe B; Willemoës, Martin; Skriver, Karen

    2015-01-15

    Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013.

  13. RNA polymerase II transcription inhibits DNA repair by photolyase in the transcribed strand of active yeast genes.

    OpenAIRE

    Livingstone-Zatchej, M; Meier, A; Suter, B.; Thoma, F

    1997-01-01

    Yeast uses nucleotide excision repair (NER) and photolyase (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs) generated by ultraviolet light. In active genes, NER preferentially repairs the transcribed strand (TS). In contrast, we recently showed that photolyase preferentially repairs the non-transcribed strands (NTS) of the URA3 and HIS3 genes in minichromosomes. To test whether photoreactivation depends on transcription, repair of CPDs was investigated in the transcriptional...

  14. The transcription factor Sox11 promotes nerve regeneration through activation of the regeneration-associated gene Sprr1a

    OpenAIRE

    Jing, Xiaotang; Wang, Ting; Huang, Shaohua; Glorioso, Joseph C.; Albers, Kathryn M.

    2011-01-01

    Factors that enhance the intrinsic growth potential of adult neurons are key players in the successful repair and regeneration of neurons following injury. Injury-induced activation of transcription factors has a central role in this process because they regulate expression of regeneration-associated genes. Sox11 is a developmentally expressed transcription factor that is significantly induced in adult neurons in response to injury. Its function in injured neurons is however undefined. Here, ...

  15. Different human TFIIIB activities direct RNA polymerase III transcription from TATA-containing and TATA-less promoters

    OpenAIRE

    Schramm, Laura; Pendergrast, P. Shannon; Sun, Yuling; Hernandez, Nouria

    2000-01-01

    Transcription initiation at RNA polymerase III promoters requires transcription factor IIIB (TFIIIB), an activity that binds to RNA polymerase III promoters, generally through protein–protein contacts with DNA binding factors, and directly recruits RNA polymerase III. Saccharomyces cerevisiae TFIIIB is a complex of three subunits, TBP, the TFIIB-related factor BRF, and the more loosely associated polypeptide β″. Although human homologs for two of the TFIIIB subunits, the TATA box–binding prot...

  16. A single, specific thymine mutation in the ComK-Binding site severely decreases binding and transcription activation by the competence transcription factor ComK of Bacillus subtilis

    NARCIS (Netherlands)

    Susanna, Kim A.; Mironczuk, Aleksandra M.; Smits, Wiep Klaas; Hamoen, Leendert W.; Kuipers, Oscar P.

    2007-01-01

    The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region. Eac

  17. Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.

    Directory of Open Access Journals (Sweden)

    Michael S Weiss

    Full Text Available BACKGROUND: Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture. METHODOLOGY/PRINCIPAL FINDINGS: TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A. CONCLUSIONS/SIGNIFICANCE: This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a

  18. The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination

    Science.gov (United States)

    Mariani, John N.; Zhang, Jingya; Liu, Jia; Sawai, Setsu; Chapouly, Candice; Horng, Sam; Kramer, Elisabeth G.; Loo, Hannah; Burlant, Natalie; Nudelman, German; Lee, Young-Min; Braun, David A.; Lu, Q. Richard; Narla, Goutham; Raine, Cedric S.; Friedman, Scott L.; Casaccia, Patrizia; John, Gareth R.

    2016-01-01

    Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination. PMID:27213272

  19. Reinstate the Damaged VEGF Signaling Pathway with VEGF-activating Transcription Factor

    Institute of Scientific and Technical Information of China (English)

    Yao-guo Yang; Heng Guan; Chang-wei Liu; Yong-jun Li

    2009-01-01

    Objective To investigate the role of vascular endothelial growth factor-activating transcriptional factor(VEGF-ATF)on the VEGF signaling pathway in diabetes mellitus.Methods Totally,20 C57BL/6 mice fed with high fat diet was induced into diabetes mellitus.Ten diabetes mellitus mice received a lower limb muscle injection with VEGF-ATF plasmid,and another ten were as control.VEGF-ATF is an engineered transcription factor designed to increase VEGF expression.Three days later,mice were sacrificed and the injected gastrocnemius was used for analysis.VEGF mRNA and protein expressions were examined by real-time PCR and ELISA respectively.VEGF receptor 2 mRNA expression was tested with RT-PCR.Phosphorylated Akt,Akt,endothelial nitric oxide synthase(eNOS),and phosphorylated eNOS were assessed by western blot.Results At 3 days post-injection,in mice with diabetes mellitus,VEGF gene transfer increased VEGF mRNA copies and VEGF protein expression in injected muscles compared with control;and reinstated the impaired VEGF signaling pathway with increasing the ratios of phosphorylated Akt/Akt and phosphorylated eNOS/eNOS.However,it did not affect the expression of VEGF receptor 2 mRNA.Conclusion Gene transfer with VEGF-ATF is able to reinstate the impaired VEGF downstream pathway,and potentially promote therapeutic angiogenesis in mice with diabetes mcllitus.

  20. SUMOylation of DEC1 Protein Regulates Its Transcriptional Activity and Enhances Its Stability

    Science.gov (United States)

    Li, Shujing; Bi, Hailian; Yang, Chunhua; Zhao, Feng; Liu, Ying; Ao, Xiang; Chang, Alan K.; Wu, Huijian

    2011-01-01

    Differentiated embryo-chondrocyte expressed gene 1 (DEC1, also known as sharp2, stra13, or BHLHB2) is a mammalian basic helix-loop-helix protein that is involved in many aspects of gene regulation through acting as a transcription factor. Changes in DEC1 expression levels have been implicated in the development of cancers. Using COS-7 cell, we showed that DEC1 can be modified by the small ubiquitin-like modifiers, SUMO1, 2 and 3. Two major SUMOylation sites (K159 and K279) were identified in the C-terminal domain of DEC1. Substitution of either K159 or K279 with arginine reduced DEC1 SUMOylation, but substitution of both K159 and K279 abolished SUMOylation, and more protein appeared to be retained in the cytoplasm compared to wild-type DEC1. The expression of DEC1 was up-regulated after serum starvation as previously reported, but at the same time, serum starvation also led to more SUMOylation of DEC1. In MCF-7 cells SUMOylation also stabilized DEC1 through inhibiting its ubiquitination. Moreover, SUMOylation of DEC1 promoted its repression of CLOCK/BMAL1-mediated transcriptional activity through recruitment of histone deacetylase1. These findings suggested that posttranslational modification of DEC1 in the form of SUMOylation may serve as a key factor that regulates the function of DEC1 in vivo. PMID:21829689

  1. Nε-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity.

    Directory of Open Access Journals (Sweden)

    Sandy Thao

    Full Text Available Evidence suggesting that eukaryotes and archaea use reversible N(ε-lysine (N(ε-Lys acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(ε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs. We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat. Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD(+-dependent Sir2 (sirtuin-like protein deacetylase (CobB deacetylated acetylated RcsB (RcsB(Ac, demonstrating that N(ε-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(ε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.

  2. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity.

    Science.gov (United States)

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N; Bradner, James E; Rabadan, Raul; Basu, Uttiya

    2015-05-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.

  3. SUMOylation of DEC1 protein regulates its transcriptional activity and enhances its stability.

    Directory of Open Access Journals (Sweden)

    Yongde Hong

    Full Text Available Differentiated embryo-chondrocyte expressed gene 1 (DEC1, also known as sharp2, stra13, or BHLHB2 is a mammalian basic helix-loop-helix protein that is involved in many aspects of gene regulation through acting as a transcription factor. Changes in DEC1 expression levels have been implicated in the development of cancers. Using COS-7 cell, we showed that DEC1 can be modified by the small ubiquitin-like modifiers, SUMO1, 2 and 3. Two major SUMOylation sites (K(159 and K(279 were identified in the C-terminal domain of DEC1. Substitution of either K(159 or K(279 with arginine reduced DEC1 SUMOylation, but substitution of both K(159 and K(279 abolished SUMOylation, and more protein appeared to be retained in the cytoplasm compared to wild-type DEC1. The expression of DEC1 was up-regulated after serum starvation as previously reported, but at the same time, serum starvation also led to more SUMOylation of DEC1. In MCF-7 cells SUMOylation also stabilized DEC1 through inhibiting its ubiquitination. Moreover, SUMOylation of DEC1 promoted its repression of CLOCK/BMAL1-mediated transcriptional activity through recruitment of histone deacetylase1. These findings suggested that posttranslational modification of DEC1 in the form of SUMOylation may serve as a key factor that regulates the function of DEC1 in vivo.

  4. Identification and characterization of jute LTR retrotransposons:: Their abundance, heterogeneity and transcriptional activity.

    Science.gov (United States)

    Ahmed, Salim; Shafiuddin, Md; Azam, Muhammad Shafiul; Islam, Md Shahidul; Ghosh, Ajit; Khan, Haseena

    2011-05-01

    Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome.

  5. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity.

    Science.gov (United States)

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N; Bradner, James E; Rabadan, Raul; Basu, Uttiya

    2015-05-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function. PMID:25957685

  6. Alteration of transcriptional networks in the entorhinal cortex after maternal immune activation and adolescent cannabinoid exposure.

    Science.gov (United States)

    Hollins, Sharon L; Zavitsanou, Katerina; Walker, Frederick Rohan; Cairns, Murray J

    2016-08-01

    Maternal immune activation (MIA) and adolescent cannabinoid exposure (ACE) have both been identified as major environmental risk factors for schizophrenia. We examined the effects of these two risk factors alone, and in combination, on gene expression during late adolescence. Pregnant rats were exposed to the viral infection mimic polyriboinosinic-polyribocytidylic acid (poly I:C) on gestational day (GD) 15. Adolescent offspring received daily injections of the cannabinoid HU210 for 14days starting on postnatal day (PND) 35. Gene expression was examined in the left entorhinal cortex (EC) using mRNA microarrays. We found prenatal treatment with poly I:C alone, or HU210 alone, produced relatively minor changes in gene expression. However, following combined treatments, offspring displayed significant changes in transcription. This dramatic and persistent alteration of transcriptional networks enriched with genes involved in neurotransmission, cellular signalling and schizophrenia, was associated with a corresponding perturbation in the expression of small non-coding microRNA (miRNA). These results suggest that a combination of environmental exposures during development leads to significant genomic remodeling that disrupts maturation of the EC and its associated circuitry with important implications as the potential antecedents of memory and learning deficits in schizophrenia and other neuropsychiatric disorders. PMID:26923065

  7. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.

    Science.gov (United States)

    Zhong, Bo; Yang, Yan; Li, Shu; Wang, Yan-Yi; Li, Ying; Diao, Feici; Lei, Caoqi; He, Xiao; Zhang, Lu; Tien, Po; Shu, Hong-Bing

    2008-10-17

    Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

  8. Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene.

    Science.gov (United States)

    Xia, Mengna; Malkaram, Sridhar A; Zempleni, Janos

    2013-11-01

    Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

  9. The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling1[OPEN

    Science.gov (United States)

    Zhou, Xin; Zhang, Zhong-Lin; Tyler, Ludmila; Yusuke, Jikumaru; Qiu, Kai; Lumba, Shelley; Desveaux, Darrell; McCourt, Peter; Sun, Tai-ping

    2016-01-01

    The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways. PMID:27255484

  10. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook;

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...

  11. Multiple steps in the regulation of transcription-factor level and activity

    NARCIS (Netherlands)

    Calkhoven, CF; Ab, G

    1996-01-01

    This review focuses on the regulation of transcription factors, many of which are DNA-binding proteins that recognize cis-regulatory elements of target genes and are the most direct regulators of gene transcription. Transcription factors serve as integration centres of the different signal-transduct

  12. Activation of Signal Transducer and Activator of Transcription 5 (STAT5) in Splenocyte Proliferation of Asthma Mice Induced by Ovalbumin

    Institute of Scientific and Technical Information of China (English)

    Guoping Li; Zhigang Liu; Peixing Ran; Jing Qiu; Nanshan Zhong

    2004-01-01

    To investigate the role of signal transducer and transcriptional activator 5 (STAT5) activated in ovalbumin (OVA)-induced splenocyte proliferation of asthma mice, an asthma mouse model was set up by intraperitoneal injection and aspiration of OVA with nebulizer. The proliferation of splenocytes isolated from the asthma mice was detected by [3H] thymidine incorporation. The phosphorytation of STAT5 was examined by Western blotting and STAT5-DNA binding was measured by electrophoretic mobility shift assay (EMSA). OVA could pronouncedly induce the splenocyte proliferation of asthma mice in a dose-dependent manner compared with control groups. Phosphorylation of STAT5 and STAT5-DNA binding were observed in splenocytes from asthma mice induced by OVA at 1 h and 3 h. These results indicated that STAT5 signal pathway played an important role in lymphocyte proliferation of asthma mice induced by OVA. Cellular & Molecular Immunology.2004;1(6):471-474.

  13. Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA

    OpenAIRE

    Kim, Jeong Hoon; Yang, Catherine K.; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is a transcriptional coactivator for nuclear receptors and enhances nuclear receptor function by the interaction with the bHLH-PAS domain (AD3) of p160 coactivators. The C-terminal activation domain (AD) of CoCoA possesses strong transactivation activity and is required for the coactivator function of CoCoA with nuclear receptors. To understand how CoCoA AD transmits its activating signal to the transcription machinery, we defined specific subregions, amino...

  14. Protolichesterinic acid derivatives: α-methylene-γ-lactones as potent dual activators of PPARγ and Nrf2 transcriptional factors.

    Science.gov (United States)

    Le Lamer, Anne-Cécile; Authier, Hélène; Rouaud, Isabelle; Coste, Agnès; Boustie, Joël; Pipy, Bernard; Gouault, Nicolas

    2014-08-15

    PPARγ and Nrf2 are important transcriptional factors involved in many signaling pathways, especially in the anti-infectious response of macrophages. Compounds bearing a Michael acceptor moiety are well known to activate such transcriptional factors, we thus evaluated the potency of α,β-unsaturated lactones synthesized using fluorous phase organic synthesis. Compounds were first screened for their cytotoxicity in order to select lactones for PPARγ and Nrf2 activation evaluation. Among them, two α-methylene-γ-lactones were identified as potent dual activators of PPARγ and Nrf2 in macrophages. PMID:25027935

  15. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    Science.gov (United States)

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.

  16. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  17. USP10 Antagonizes c-Myc Transcriptional Activation through SIRT6 Stabilization to Suppress Tumor Formation

    Directory of Open Access Journals (Sweden)

    Zhenghong Lin

    2013-12-01

    Full Text Available The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.

  18. AhR transcription serum activity of Inuit´s across Greenlandic districts

    DEFF Research Database (Denmark)

    Long, Manhai; Deutch, Bente; Bonefeld-Jørgensen, Eva Cecilie

    2007-01-01

    Background: Human exposure to lipophilic persistent organic pollutants (POPs) including Human exposure to lipophilic persistent organic pollutants (POPs) including polychlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs), polychlorinated biphenyls (PCBs) and organochlorine pesticide is ubiquitous....... The individual is exposed to a complex mixture of POPs being life-long beginning during critical developmental windows. Exposure to POPs elicits a number of species- and tissue-specific toxic responses, many of which involve the aryl hydrocarbon receptor (AhR). The aim of this study was to compare the actual...... level of integrated AhR transcriptional activity in the lipophilic serum fraction containing the actual POP mixture among Inuit's from different districts in Greenland, and to evaluate whether the AhR transactivity is correlated to the bio-accumulated POPs and /or lifestyle factors. Methods: The study...

  19. KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification.

    Science.gov (United States)

    Tetreault, Marie-Pier; Weinblatt, Daniel; Shaverdashvili, Khvaramze; Yang, Yizeng; Katz, Jonathan P

    2016-05-17

    Epithelial differentiation and stratification are essential for normal homeostasis, and disruption of these processes leads to both injury and cancer. The zinc-finger transciption factor KLF4 is a key driver of epithelial differentiation, yet the mechanisms and targets by which KLF4 controls differentiation are not well understood. Here, we define WNT5A, a non-canonical Wnt ligand implicated in epithelial differentiation, repair, and cancer, as a direct transcriptional target that is activated by KLF4 in squamous epithelial cells. Further, we demonstrate functionally that WNT5A mediates KLF4 control of epithelial differentiation and stratification, as treatment of keratinocytes with WNT5A rescues defective epithelial stratification resulting from KLF4 loss. Finally, we show that the small GTPase CDC42 is regulated by KLF4 in a WNT5A dependent manner. As such, we delineate a novel pathway for epithelial differentiation and stratification and define potential therapeutic targets for epithelial diseases.

  20. Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo

    DEFF Research Database (Denmark)

    Nudelman, Aaron Samuel; DiRocco, Derek P; Lambert, Talley J;

    2010-01-01

    expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus......, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h...

  1. A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    张宁; 徐永健; 张珍祥; 熊维宁

    2003-01-01

    To determine the feasibility of a nonradioactive electrophoresis mobility shift assay fordetecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus tar-get sequence of NF-κB were labled with DIG by terminal transferase. After nuclear protein stimula-ted with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDT C)electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, theywere electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemilu-minescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded spe-cifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method andthe activity of NF-κB in PMA group was more than that in PMA+PDTC group. It is suggestedthat detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, whichcan be performed in ordinary laboratories.

  2. A Global Genomic and Genetic Strategy to Predict Pathway Activation of Xenobiotic Responsive Transcription Factors in the Mouse Liver

    Science.gov (United States)

    Many drugs and environmentally-relevant chemicals activate xenobiotic-responsive transcription factors(TF). Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening. Starting with a large compendium of Affymet...

  3. The transcriptional activation program of human neutrophils in skin lesions supports their important role in wound healing

    DEFF Research Database (Denmark)

    Theilgaard-Monch, K.; Knudsen, Steen; Follin, P.;

    2004-01-01

    . Notably, the up-regulation of genes, which activate lysosomal proteases, indicate a priming of skin lesion-PMNs for degradation of phagocytosed material. These findings demonstrate that migration of PMNs to skin lesions induces a transcriptional activation program, which regulates cellular fate...

  4. Mutational analysis of the activator of late transcription, Alt , in the lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Hammer, Karin

    2007-01-01

    An activator protein, Alt, synthesized during the early state of lytic infection is required for transcription of the late operon in the lactococcal phage TP901-1. In order to identify amino acid residues in the Alt protein required for activation of the TP901-1 late promoter, Plate, hydroxylamin...

  5. Retrotransposon and gene activation in wheat in response to mycotoxigenic and non-mycotoxigenic-associated Fusarium stress

    DEFF Research Database (Denmark)

    Ansari, Khairul Islam; Walter, Stephanie; Brennan, Josephine M.;

    2007-01-01

    zipper protein transcription factor (bZIP), retrotransposon-like homologs and genes of unknown function in the roots of wheat cultivars CM82036 and Remus. Fusarium head blight (FHB) studies using Fusarium graminearum and its trichothecene-minus (Tri5(-)) mutant derivative and adult plant DON tests showed...... that these transcripts were responsive to both mycotoxigenic- and non-mycotoxigenic-associated Fusarium stress. In tests using the parents 'CyCM82036', 'Remus' and 14 double-haploid progeny that segregated for quantitative trait locus (QTL) Fhb1 on chromosome 3BS (syn. Qfhs.ndsu-3BS) (from 'CyCM82036' that confers DON......-regulated wheat bZIP (wLIP19) maps to group 1 chromosomes suggests that wheat bZIP may participate in defence response cascades associated with Fhb1 and that there is a cross-talk between biotic and abiotic stress signalling pathways....

  6. Mechanisms of transcriptional activation of the mouse claudin-5 promoter by estrogen receptor alpha and beta.

    Science.gov (United States)

    Burek, Malgorzata; Steinberg, Katrin; Förster, Carola Y

    2014-07-01

    Claudin-5 is an integral membrane protein and a critical component of endothelial tight junctions that control paracellular permeability. Claudin-5 is expressed at high levels in the brain vascular endothelium. Estrogens have multiple effects on vascular physiology and function. The biological actions of estrogens are mediated by two different estrogen receptor (ER) subtypes, ER alpha and ER beta. Estrogens have beneficial effects in several vascular disorders. Recently we have cloned and characterized a murine claudin-5 promoter and demonstrated 17beta-estradiol (E2)-mediated regulation of claudin-5 in brain and heart microvascular endothelium on promoter, mRNA and protein level. Sequence analysis revealed a putative estrogen response element (ERE) and a putative Sp1 transcription factor binding site in the claudin-5 promoter. The aim of the present study was to further characterize the estrogen-responsive elements of claudin-5 promoter. First, we introduced point mutations in ERE or Sp1 site in -500/+111 or in Sp1 site of -268/+111 claudin-5 promoter construct, respectively. Basal and E2-mediated transcriptional activation of mutated constructs was abrogated in the luciferase reporter gene assay. Next, we examined whether estrogen receptor subtypes bind to the claudin-5 promoter region. For this purpose we performed chromatin immunoprecipitation assays using anti-estrogen receptor antibodies and cellular lysates of E2-treated endothelial cells followed by quantitative PCR analysis. We show enrichment of claudin-5 promoter fragments containing the ERE- and Sp1-binding site in immunoprecipitates after E2 treatment. Finally, in a gel mobility shift assay, we demonstrated DNA-protein interaction of both ER subtypes at ERE. In summary, this study provides evidence that both a non-consensus ERE and a Sp1 site in the claudin-5 promoter are functional and necessary for the basal and E2-mediated activation of the promoter.

  7. Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma

    Directory of Open Access Journals (Sweden)

    Lazarus Ross

    2005-06-01

    Full Text Available Abstract Background Identifying genetic determinants for lung function is important in providing insight into the pathophysiology of asthma. Signal transducer and activator of transcription 3 is a transcription factor latent in the cytoplasm; the gene (STAT3 is activated by a wide range of cytokines, and may play a role in lung development and asthma pathogenesis. Methods We genotyped six single nucleotide polymorphisms (SNPs in the STAT3 gene in a cohort of 401 Caucasian adult asthmatics. The associations between each SNP and forced expiratory volume in 1 second (FEV1, as a percent of predicted, at the baseline exam were tested using multiple linear regression models. Longitudinal analyses involving repeated measures of FEV1 were conducted with mixed linear models. Haplotype analyses were conducted using imputed haplotypes. We completed a second association study by genotyping the same six polymorphisms in a cohort of 652 Caucasian children with asthma. Results We found that three polymorphisms were significantly associated with baseline FEV1: homozygotes for the minor alleles of each polymorphism had lower FEV1 than homozygotes for the major alleles. Moreover, these associations persisted when we performed an analysis on repeated measures of FEV1 over 8 weeks. A haplotypic analysis based on the six polymorphisms indicated that two haplotypes were associated with baseline FEV1. Among the childhood asthmatics, one polymorphism was associated with both baseline FEV1 and the repeated measures of FEV1 over 4 years. Conclusion Our results indicate that genetic variants in STAT3, independent of asthma treatment, are determinants of FEV1 in both adults and children with asthma, and suggest that STAT3 may participate in inflammatory pathways that have an impact on level of lung function.

  8. Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid.

    Science.gov (United States)

    Zhang, Dongxian; Lee, Brian; Nutter, Anthony; Song, Paul; Dolatabadi, Nima; Parker, James; Sanz-Blasco, Sara; Newmeyer, Traci; Ambasudhan, Rajesh; McKercher, Scott R; Masliah, Eliezer; Lipton, Stuart A

    2015-06-01

    Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2). PMID:25692407

  9. Quercetin represses apolipoprotein B expression by inhibiting the transcriptional activity of C/EBPβ.

    Directory of Open Access Journals (Sweden)

    Makoto Shimizu

    Full Text Available Quercetin is one of the most abundant polyphenolic flavonoids found in fruits and vegetables and has anti-oxidative and anti-obesity effects. Because the small intestine is a major absorptive organ of dietary nutrients, it is likely that highly concentrated food constituents, including polyphenols, are present in the small intestinal epithelial cells, suggesting that food factors may have a profound effect in this tissue. To identify novel targets of quercetin in the intestinal enterocytes, mRNA profiling using human intestinal epithelial Caco-2 cells was performed. We found that mRNA levels of some apolipoproteins, particularly apolipoprotein B (apoB, are downregulated in the presence of quercetin. On the exposure of Caco-2 cells to quercetin, both mRNA and protein levels of apoB were decreased. Promoter analysis of the human apoB revealed that quercetin response element is localized at the 5'-proximal promoter region, which contains a conserved CCAAT enhancer-binding protein (C/EBP-response element. We found that quercetin reduces the promoter activity of apoB, driven by the enforced expression of C/EBPβ. Quercetin had no effect on either mRNA or protein levels of C/EBPβ. In contrast, we found that quercetin inhibits the transcriptional activity of C/EBPβ but not its recruitment to the apoB promoter. On the exposure of Caco-2 cells to quercetin 3-O-glucuronide, which is in a cell-impermeable form, no notable change in apoB mRNA was observed, suggesting an intracellular action of quercetin. In vitro interaction experiments using quercetin-conjugated beads revealed that quercetin binds to C/EBPβ. Our results describe a novel regulatory mechanism of transcription of apolipoprotein genes by quercetin in the intestinal enterocytes.

  10. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    Energy Technology Data Exchange (ETDEWEB)

    Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States); Edelman, Jeffrey L.; Brooks, Patricia A. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  11. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    International Nuclear Information System (INIS)

    Highlights: ► siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. ► siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. ► Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (−325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  12. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

    Science.gov (United States)

    Mukhopadhyay, C. K.; Mazumder, B.; Fox, P. L.

    2000-01-01

    A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these

  13. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2015-01-30

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  14. Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival

    Directory of Open Access Journals (Sweden)

    Hsieh Fu-Chuan

    2008-10-01

    Full Text Available Abstract Background Constitutive activation of signal transducer and activator of transcription 3 (Stat3 signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer. Results We found that elevated Stat3 phosphorylation in 19 of 100 (19% bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC. The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin and a cell cycle regulating gene (cyclin D1 was associated with the cell growth inhibition and apoptosis. Conclusion These results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

  15. Intrinsic HER4/4ICD transcriptional activation domains are required for STAT5A activated gene expression.

    Science.gov (United States)

    Han, Wen; Sfondouris, Mary E; Semmes, Eleanor C; Meyer, Alicia M; Jones, Frank E

    2016-10-30

    The epidermal growth factor receptor family member HER4 undergoes proteolytic processing at the cell surface to release the HER4 intracellular domain (4ICD) nuclear protein. Interestingly, 4ICD directly interacts with STAT5 and functions as an obligate STAT5 nuclear chaperone. Once in the nucleus 4ICD binds with STAT5 at STAT5 target genes, dramatically potentiating STAT5 transcriptional activation. These observations raise the possibility that 4ICD directly coactivates STAT5 gene expression. Using both yeast and mammalian transactivation reporter assays, we performed truncations of 4ICD fused to a GAL4 DNA binding domain and identified two independent 4ICD transactivation domains located between residues 1022 and 1090 (TAD1) and 1192 and 1225 (TAD2). The ability of the 4ICD DNA binding domain fusions to transactivate reporter gene expression required deletion of the intrinsic tyrosine kinase domain. In addition, we identified the 4ICD carboxyl terminal TVV residues, a PDZ domain binding motif (PDZ-DBM), as a potent transcriptional repressor. The transactivation activity of the HER4 carboxyl terminal domain lacking the tyrosine kinase (CTD) was significantly lower than similar EGFR or HER2 CTD. However, deletion of the HER4 CTD PDZ-DBM enhanced HER4 CTD transactivation to levels equivalent to the EGFR and HER2 CTDs. To determine if 4ICD TAD1 and TAD2 have a physiologically relevant role in STAT5 transactivation, we coexpressed 4ICD or 4ICD lacking TAD2 or both TAD1 and TAD2 with STAT5 in a luciferase reporter assay. Our results demonstrate that each 4ICD TAD contributes additively to STAT5A transactivation and the ability of STAT5A to transactivate the β-casein promoter requires the 4ICD TADs. Taken together, published data and our current results demonstrate that both 4ICD nuclear chaperone and intrinsic coactivation activities are essential for STAT5 regulated gene expression. PMID:27502417

  16. Resveratrol enhances antitumor activity of TRAIL in prostate cancer xenografts through activation of FOXO transcription factor.

    Directory of Open Access Journals (Sweden)

    Suthakar Ganapathy

    Full Text Available BACKGROUND: Resveratrol (3, 4', 5 tri-hydroxystilbene, a naturally occurring polyphenol, exhibits anti-inflammatory, antioxidant, cardioprotective and antitumor activities. We have recently shown that resveratrol can enhance the apoptosis-inducing potential of TRAIL in prostate cancer cells through multiple mechanisms in vitro. Therefore, the present study was designed to validate whether resveratrol can enhance the apoptosis-inducing potential of TRAIL in a xenograft model of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: Resveratrol and TRAIL alone inhibited growth of PC-3 xenografts in nude mice by inhibiting tumor cell proliferation (PCNA and Ki67 staining and inducing apoptosis (TUNEL staining. The combination of resveratrol and TRAIL was more effective in inhibiting tumor growth than single agent alone. In xenografted tumors, resveratrol upregulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and p27(/KIP1, and inhibited the expression of Bcl-2 and cyclin D1. Treatment of mice with resveratrol and TRAIL alone inhibited angiogenesis (as demonstrated by reduced number of blood vessels, and VEGF and VEGFR2 positive cells and markers of metastasis (MMP-2 and MMP-9. The combination of resveratrol with TRAIL further inhibited number of blood vessels in tumors, and circulating endothelial growth factor receptor 2-positive endothelial cells than single agent alone. Furthermore, resveratrol inhibited the cytoplasmic phosphorylation of FKHRL1 resulting in its enhanced activation as demonstrated by increased DNA binding activity. CONCLUSIONS/SIGNIFICANCE: These data suggest that resveratrol can enhance the apoptosis-inducing potential of TRAIL by activating FKHRL1 and its target genes. The ability of resveratrol to inhibit tumor growth, metastasis and angiogenesis, and enhance the therapeutic potential of TRAIL suggests that resveratrol alone or in combination with TRAIL can be used for the management of prostate cancer.

  17. BAF Complex Is Closely Related to and Interacts with NF1/CTF and RNA Polymerase Ⅱ in Gene Transcriptional Activation

    Institute of Scientific and Technical Information of China (English)

    Li-Hui ZHAO; Xue-Qing BA; Xiao-Guang WANG; Xiao-Juan ZHU; Li WANG; Xian-Lu ZENG

    2005-01-01

    Brg- or hBrm-associated factor (BAF) complexes, a chromatin-remodeling complex family of mammalian cells, facilitate transcriptional activity by remodeling nucleosome structure. Brg 1 is the core subunit of Brg-associated factor complexes. In the present study, we investigated the spatial relationship between Brg1 and nuclear factor 1 (NF1/CTF) and RNA polymerase Ⅱ (RNAP Ⅱ) upon gene transcriptional activation in vivo by employing immuno-gold labeling. The data showed that Brg1 was closely co-localized with NF1/CTF and RNAP Ⅱ in HeLa cells. Moreover, the co-immunoprecipitation assay further revealed that Brg1 can be isolated together with NF1/CTF and RNAP Ⅱ in the ConA-stimulated, but not the resting,T lymphocyte. The combined results suggested that BAF complexes can interact with NF1/CTF and RNAP Ⅱ, and this interaction is closely dependent on the activation of gene transcription.

  18. CMYB1 Encoding a MYB Transcriptional Activator Is Involved in Abiotic Stress and Circadian Rhythm in Rice

    Directory of Open Access Journals (Sweden)

    Min Duan

    2014-01-01

    Full Text Available Through analysis of cold-induced transcriptome, a novel gene encoding a putative MYB transcription factor was isolated and designated Cold induced MYB 1 (CMYB1. Tissue-specific gene expression analysis revealed that CMYB1 was highly expressed in rice stems and nodes. qRT-PCR assay indicated that CMYB1 was dramatically induced by cold stress (>100-folds and induced by exogenous ABA and osmotic stress. Interestingly, CMYB1 showed rhythmic expression profile in rice leaves at different developmental stages. Subcellular localization assay suggested that CMYB1-GFP (green fluorescent protein fusion protein was localized in the nuclei. Moreover, CMYB1 exhibited the transcriptional activation activity when transiently expressed in rice protoplast cells. Taken together, CMYB1 probably functions as a transcriptional activator in mediating stress and rhythm responsive gene expression in rice.

  19. Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

    Directory of Open Access Journals (Sweden)

    Irma Eloisa Monroy-Muñoz

    2015-01-01

    Full Text Available Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD using the High Resolution Melting (HRM method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up.

  20. Transcriptional activation by pRB and its coordination with SWI/SNF recruitment.

    Science.gov (United States)

    Flowers, Stephen; Beck, George R; Moran, Elizabeth

    2010-11-01

    A central question in cancer biology is why most tumor susceptibility genes are linked with only limited types of cancer. Human germ-line mutation of the retinoblastoma susceptibility gene Rb1 is closely linked with just retinoblastoma and osteosarcoma, although the gene is universally expressed. Functional analysis of pRB and its close relatives, p107 and p130, has largely focused on their roles in repression of proliferation across all tissue types, but genetic evidence indicates an active requirement for pRB in osteoblast differentiation that correlates more directly with osteosarcoma susceptibility. Still, potential promoter targets of pRB and its role in normally differentiating osteoblasts remain insufficiently characterized. Here, an early marker of osteoblast differentiation, alkaline phosphatase, is identified as a direct promoter activation target of pRB. One role of pRB on this promoter is to displace the histone lysine demethylase KDM5A, thereby favoring trimethylation of H3K4, a promoter activation mark. A major new aspect of pRB-mediated transcriptional activation revealed in this promoter analysis is its role in recruitment of an activating SWI/SNF chromatin-remodeling complex. SWI/SNF is a critical coordinator of tissue-specific gene expression. In osteoblasts, SWI/SNF complexes containing the BRM ATPase repress osteoblast-specific genes to maintain the precursor state, whereas the alternative ATPase BRG1 distinguishes an activating SWI/SNF complex necessary for RNA polymerase-II recruitment. A switch from BRM to BRG1 on the alkaline phosphatase promoter marks the onset of differentiation and is accomplished in a precise two-step mechanism. Dissociation of BRM-containing SWI/SNF depends on p300, and association of BRG1-containing SWI/SNF depends on pRB.

  1. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB.

    Science.gov (United States)

    Yu, Zhiyuan; Zhang, Wenzheng; Kone, Bruce C

    2002-01-01

    Prolific generation of NO by inducible nitric oxide synthase (iNOS) can cause unintended injury to host cells during glomerulonephritis and other inflammatory diseases. While much is known about the mechanisms of iNOS induction, few transcriptional repressors have been found. We explored the role of signal transducers and activators of transcription 3 (STAT3) proteins in interleukin (IL)-1beta- and lipopolysaccharide (LPS)+interferon (IFN)-gamma-mediated iNOS induction in murine mesangial cells. Both stimuli induced rapid phosphorylation of STAT3 and sequence-specific STAT3 DNA-binding activity. Supershift assays with a STAT3 element probe demonstrated that nuclear factor kappaB (NF-kappaB) p65 and p50 complexed with STAT3 in the DNA-protein complex. The direct interaction of STAT3 and NF-kappaB p65 was verified in vivo by co-immunoprecipitation and in vitro by pull-down assays with glutathione S-transferase-NF-kappaB p65 fusion protein and in vitro -translated STAT3alpha. Overexpression of STAT3 dramatically inhibited IL-1beta- or LPS+IFN-gamma-mediated induction of iNOS promoter-luciferase constructs that contained the wild-type iNOS promoter or ones harbouring mutated STAT-binding elements. In tests of indirect inhibitory effects of STAT3, overexpression of STAT3 dramatically inhibited the activity of an NF-kappaB-dependent promoter devoid of STAT-binding elements without affecting NF-kappaB DNA-binding activity. Thus STAT3, via direct interactions with NF-kappaB p65, serves as a dominant-negative inhibitor of NF-kappaB activity to suppress indirectly cytokine induction of the iNOS promoter in mesangial cells. These results provide a new model for the termination of NO production by activated iNOS following exposure to pro-inflammatory stimuli. PMID:12057007

  2. Transcriptional dysregulation of inflammatory/immune pathways after active vaccination against Huntington's disease.

    Science.gov (United States)

    Ramsingh, Arlene I; Manley, Kevin; Rong, Yinghui; Reilly, Andrew; Messer, Anne

    2015-11-01

    Immunotherapy, both active and passive, is increasingly recognized as a powerful approach to a wide range of diseases, including Alzheimer's and Parkinson's. Huntington's disease (HD), an autosomal dominant disorder triggered by misfolding of huntingtin (HTT) protein with an expanded polyglutamine tract, could also benefit from this approach. Individuals can be identified genetically at the earliest stages of disease, and there may be particular benefits to a therapy that can target peripheral tissues in addition to brain. In this active vaccination study, we first examined safety and immunogenicity for a broad series of peptide, protein and DNA plasmid immunization protocols, using fragment (R6/1), and knock-in (zQ175) models. No safety issues were found. The strongest and most uniform immune response was to a combination of three non-overlapping HTT Exon1 coded peptides, conjugated to KLH, delivered with alum adjuvant. An N586-82Q plasmid, delivered via gene gun, also showed ELISA responses, mainly in the zQ175 strain, but with more variability, and less robust responses in HD compared with wild-type controls. Transcriptome profiling of spleens from the triple peptide-immunized cohort showed substantial HD-specific differences including differential activation of genes associated with innate immune responses, absence of negative feedback control of gene expression by regulators, a temporal dysregulation of innate immune responses and transcriptional repression of genes associated with memory T cell responses. These studies highlight critical issues for immunotherapy and HD disease management in general. PMID:26307082

  3. Transcriptional pathways in cPGI2-induced adipocyte progenitor activation for browning

    Directory of Open Access Journals (Sweden)

    Irem eBayindir

    2015-08-01

    Full Text Available De novo formation of beige/brite adipocytes from progenitor cells contributes to the thermogenic adaptation of adipose tissue and holds great potential for the therapeutic remodeling of fat as a treatment for obesity. Despite the recent identification of several factors regulating browning of white fat, there is a lack of physiological cell models for the mechanistic investigation of progenitor-mediated beige/brite differentiation. We have previously revealed prostacyclin (PGI2 as one of the few known endogenous extracellular mediators promoting de novo beige/brite formation by relaying beta-adrenergic stimulation to the progenitor level. Here we present a cell model based on murine primary progenitor cells defined by markers previously shown to be relevant for in vivo browning, including a simplified isolation procedure. We demonstrate the specific and broad induction of thermogenic gene expression by PGI2 signaling in the absence of lineage conversion, and reveal the previously unidentified nuclear relocalization of the Ucp1 gene locus in association with transcriptional activation. By profiling the time course of the progenitor response we show that PGI2 signaling promoted progenitor cell activation through cell cycle and adhesion pathways prior to metabolic maturation towards an oxidative cell phenotype. Our results highlight the importance of core progenitor activation pathways for the recruitment of thermogenic cells and provide a resource for further mechanistic investigation.

  4. Non-additive transcriptional profiles underlie dikaryotic superiority in Pleurotus ostreatus laccase activity.

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    Raúl Castanera

    Full Text Available BACKGROUND: The basidiomycete Pleurotus ostreatus is an efficient producer of laccases, a group of enzymes appreciated for their use in multiple industrial processes. The aim of this study was to reveal the molecular basis of the superiority of laccase production by dikaryotic strains compared to their parental monokaryons. METHODOLOGY/PRINCIPAL FINDINGS: We bred and studied a set of dikaryotic strains starting from a meiotic population of monokaryons. We then completely characterised the laccase allelic composition, the laccase gene expression and activity profiles in the dikaryotic strain N001, in two of its meiotic full-sib monokaryons and in the dikaryon formed from their mating. CONCLUSIONS/SIGNIFICANCE: Our results suggested that the dikaryotic superiority observed in laccase activity was due to non-additive transcriptional increases in lacc6 and lacc10 genes. Furthermore, the expression of these genes was divergent in glucose- vs. lignocellulose-supplemented media and was highly correlated to the detected extracellular laccase activity. Moreover, the expression profile of lacc2 in the dikaryotic strains was affected by its allelic composition, indicating a putative single locus heterozygous advantage.

  5. Arabidopsis resistance protein SNC1 activates immune responses through association with a transcriptional corepressor

    Science.gov (United States)

    Zhu, Zhaohai; Xu, Fang; Zhang, Yaxi; Cheng, Yu Ti; Wiermer, Marcel; Li, Xin; Zhang, Yuelin

    2010-01-01

    In both plants and animals, nucleotide-binding (NB) domain and leucine-rich repeat (LRR)-containing proteins (NLR) function as sensors of pathogen-derived molecules and trigger immune responses. Although NLR resistance (R) proteins were first reported as plant immune receptors more than 15 years ago, how these proteins activate downstream defense responses is still unclear. Here we report that the Toll-like/interleukin-1 receptor (TIR)-NB-LRR R protein, suppressor of npr1-1, constitutive 1 (SNC1) functions through its associated protein, Topless-related 1 (TPR1). Knocking out TPR1 and its close homologs compromises immunity mediated by SNC1 and several other TIR-NB-LRR–type R proteins, whereas overexpression of TPR1 constitutively activates SNC1-mediated immune responses. TPR1 functions as a transcriptional corepressor and associates with histone deacetylase 19 in vivo. Among the target genes of TPR1 are Defense no Death 1 (DND1) and Defense no Death 2 (DND2), two known negative regulators of immunity that are repressed during pathogen infection, suggesting that TPR1 activates R protein-mediated immune responses through repression of negative regulators. PMID:20647385

  6. Molecular imaging of nuclear factor-Y transcriptional activity maps proliferation sites in live animals.

    Science.gov (United States)

    Goeman, Frauke; Manni, Isabella; Artuso, Simona; Ramachandran, Balaji; Toietta, Gabriele; Bossi, Gianluca; Rando, Gianpaolo; Cencioni, Chiara; Germoni, Sabrina; Straino, Stefania; Capogrossi, Maurizio C; Bacchetti, Silvia; Maggi, Adriana; Sacchi, Ada; Ciana, Paolo; Piaggio, Giulia

    2012-04-01

    In vivo imaging involving the use of genetically engineered animals is an innovative powerful tool for the noninvasive assessment of the molecular and cellular events that are often targets of therapy. On the basis of the knowledge that the activity of the nuclear factor-Y (NF-Y) transcription factor is restricted in vitro to proliferating cells, we have generated a transgenic reporter mouse, called MITO-Luc (for mitosis-luciferase), in which an NF-Y-dependent promoter controls luciferase expression. In these mice, bioluminescence imaging of NF-Y activity visualizes areas of physiological cell proliferation and regeneration during response to injury. Using this tool, we highlight for the first time a role of NF-Y activity on hepatocyte proliferation during liver regeneration. MITO-Luc reporter mice should facilitate investigations into the involvement of genes in cell proliferation and provide a useful model for studying aberrant proliferation in disease pathogenesis. They should be also useful in the development of new anti/proproliferative drugs and assessment of their efficacy and side effects on nontarget tissues. PMID:22379106

  7. Characterization of transcriptional activation and DNA-binding functions in the hinge region of the vitamin D receptor.

    Science.gov (United States)

    Shaffer, Paul L; McDonnell, Donald P; Gewirth, Daniel T

    2005-02-22

    The vitamin D receptor (VDR) is a ligand-responsive transcription factor that forms active, heterodimeric complexes with the 9-cis retinoic acid receptor (RXR) on vitamin D response elements (VDREs). Both proteins consist of an N-terminal DNA-binding domain, a C-terminal ligand-binding domain, and an intervening hinge region. The length requirements of the hinge for both transcriptional regulation and DNA binding have not been studied to date for any member of the steroid hormone superfamily. We have generated a series of internal deletion mutants of the VDR hinge and found that deletion of as few as five amino acids from the C-terminus of the hinge significantly reduces transcriptional activation in vivo. Replacing deleted residues in the C-terminus of the hinge with alanines restored activity, indicating that this section of the hinge acts as a sequence-independent spacer. The hinge region of VDR forms a long helix, and the geometric consequences of this structure may explain the requirement of the hinge region for transcriptional activity. Interestingly, all of the deletion mutants, even those that do not activate transcription, bind VDREs with equal and high affinity, indicating that the defect in these mutants is not their ability to bind VDREs. In contrast to VDR, constructs of RXR containing deletions of up to 14 amino acids in the hinge region exhibit near wild-type transcriptional activity. The ability to delete more of the RXR hinge may be related to the additional plasticity required by its role as the common heterodimer partner for nuclear receptors on differing DNA elements.

  8. The Runx transcriptional co-activator, CBFβ, is essential for invasion of breast cancer cells

    Directory of Open Access Journals (Sweden)

    Lopez-Camacho Cesar

    2010-06-01

    Full Text Available Abstract Background The transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBFβ is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBFβ is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBFβ-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBFβ. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBFβ expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBFβ. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis. Results We show that CBFβ is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBFβ contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBFβ is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBFβ is recruited to both the Osteopontin and the Galectin-3 promoters. Conclusions CBFβ is expressed in metastatic breast cancer cells and is essential for cell invasion. CBFβ is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBFβ is essential for invasion, not all Runx2-target genes require CBFβ. We conclude that CBFβ is required for a subset of Runx2-target genes

  9. Unique transcriptional profile of sustained ligand-activated preconditioning in pre- and post-ischemic myocardium.

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    Kevin J Ashton

    Full Text Available BACKGROUND: Opioidergic SLP (sustained ligand-activated preconditioning induced by 3-5 days of opioid receptor (OR agonism induces persistent protection against ischemia-reperfusion (I-R injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium. METHODOLOGY/PRINCIPAL FINDINGS: Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%. Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des, natriuretic peptides (Nppa,Nppb and stress-signaling elements (Csda,Ptgds. Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3, cytokines (Il1b,Il6,Tnf and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3, together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1 and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid. Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia, which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2 and other forms of stress (Xirp1,Ankrd1,Clu, and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1 and Txnip. CONCLUSIONS: Protection via SLP is associated with transcriptional repression of inflammation/immunity, up

  10. Molecular recognition: monomer of the yeast transcriptional activator GCN4 recognizes its dimer DNA binding target sites specifically

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    It is widely believed that dimerization is a requirement for the yeast transcriptional activator GCN4 to recognize its specific DNA target sites. We used the basic region (226-252) of the yeast transcriptional activator GCN4, as both a monomeric peptide and a disulfide-linked dimer to investigate the interaction of the peptides with the DNA target sites AP-1 and CRE. CD and ITC experiments indicate that although the monomeric peptide GCN4-M has a weaker affinity with the DNA relative to the disulfide-linked dimer peptide GCN4-D, it recognizes AP-1 and CRE target sites specifically.

  11. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    Science.gov (United States)

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  12. HIV-1 trans-activator of transcription substitutes for oxidative signaling in activation-induced T cell death.

    Science.gov (United States)

    Gülow, Karsten; Kaminski, Marcin; Darvas, Katalin; Süss, Dorothee; Li-Weber, Min; Krammer, Peter H

    2005-05-01

    Termination of an immune response requires elimination of activated T lymphocytes by activation-induced cell death (AICD). In AICD, CD95 (Apo-1/Fas) ligand (L) triggers apoptosis of CD95-positive activated T lymphocytes. In AIDS patients, AICD is strongly enhanced and accelerated. We and others have previously shown that HIV-1 trans-activator of transcription (HIV-1 Tat) sensitizes T cells toward CD95-mediated apoptosis and up-regulates CD95L expression by affecting the cellular redox balance. In this study, we show that it is hydrogen peroxide (H(2)O(2)) that functions as an essential second messenger in TCR signaling. The H(2)O(2) signal combined with simultaneous calcium (Ca(2+)) influx into the cytosol constitutes the minimal requirement for induction of CD95L expression. Either signal alone is insufficient. We further show that HIV-1 Tat interferes with TCR signaling and induces a H(2)O(2) signal. H(2)O(2) generated by HIV-1 Tat combines with CD4-dependent calcium influx and causes massive T cell apoptosis. Thus, our data provide an explanation for CD4(+) T lymphocyte depletion during progression of AIDS.

  13. The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Vidal M.S.

    2002-01-01

    Full Text Available Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22. Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

  14. Ribbon regulates morphogenesis of the Drosophila embryonic salivary gland through transcriptional activation and repression.

    Science.gov (United States)

    Loganathan, Rajprasad; Lee, Joslynn S; Wells, Michael B; Grevengoed, Elizabeth; Slattery, Matthew; Andrew, Deborah J

    2016-01-01

    Transcription factors affect spatiotemporal patterns of gene expression often regulating multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape/volume increases during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the changes in cell shape/volume in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib, we performed ChIP-seq analysis in embryos driving expression of GFP-tagged Rib specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites bound by Rib likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell growth and tissue shape in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that autoregulation of rib expression may be a key component of the SG morphogenetic gene network.

  15. RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13.

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    Ila Datar

    Full Text Available Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP

  16. Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation

    Science.gov (United States)

    Fuda, Nicholas J.; Mahat, Dig B.; Core, Leighton J.; Guertin, Michael J.

    2016-01-01

    The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes. PMID:27492368

  17. Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcriptional activator modulating reactive oxygen species accumulation and cell death.

    Science.gov (United States)

    Niu, Fangfang; Wang, Chen; Yan, Jingli; Guo, Xiaohua; Wu, Feifei; Yang, Bo; Deyholos, Michael K; Jiang, Yuan-Qing

    2016-09-01

    NAC transcription factors (TFs) are plant-specific and play important roles in development, responses to biotic and abiotic cues and hormone signaling. So far, only a few NAC genes have been reported to regulate cell death. In this study, we identified and characterized a NAC55 gene isolated from oilseed rape (Brassica napus L.). BnaNAC55 responds to multiple stresses, including cold, heat, abscisic acid (ABA), jasmonic acid (JA) and a necrotrophic fungal pathogen Sclerotinia sclerotiorum. BnaNAC55 has transactivation activity and is located in the nucleus. BnaNAC55 is able to form homodimers in planta. Unlike ANAC055, full-length BnaNAC55, but not either the N-terminal NAC domain or C-terminal regulatory domain, induces ROS accumulation and hypersensitive response (HR)-like cell death when expressed both in oilseed rape protoplasts and Nicotiana benthamiana. Furthermore, BnaNAC55 expression causes obvious nuclear DNA fragmentation. Moreover, quantitative reverse transcription PCR (qRT-PCR) analysis identified that the expression levels of multiple genes regulating ROS production and scavenging, defense response as well as senescence are significantly induced. Using a dual luciferase reporter assay, we further confirm that BnaNAC55 could activate the expression of a few ROS and defense-related gene expression. Taken together, our work has identified a novel NAC TF from oilseed rape that modulates ROS accumulation and cell death. PMID:27312204

  18. Site-specific phosphorylation regulates the transcriptive activity of vesicular stomatitis virus NS protein.

    OpenAIRE

    C. H. Hsu; Morgan, E M; Kingsbury, D. W.

    1982-01-01

    In vitro transcription by vesicular stomatitis virus nucleocapsids is inhibited by enzymatic dephosphorylation of the NS protein. We provide evidence that specific, partial dephosphorylation of NS molecules is the only detectable change in nucleocapsids treated with bacterial alkaline phosphatase under conditions that prevent the action of adventitious protease. Dephosphorylation appeared to affect only the rate of transcription; there were no changes in sedimentation rates of transcripts. To...

  19. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    Science.gov (United States)

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background. PMID:23630316

  20. Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense.

    Science.gov (United States)

    Lai, Zhibing; Li, Ying; Wang, Fei; Cheng, Yuan; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2011-10-01

    Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif-containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens.

  1. Drosophila SAF-B links the nuclear matrix, chromosomes, and transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Catalina Alfonso-Parra

    Full Text Available Induction of gene expression is correlated with alterations in nuclear organization, including proximity to other active genes, to the nuclear cortex, and to cytologically distinct domains of the nucleus. Chromosomes are tethered to the insoluble nuclear scaffold/matrix through interaction with Scaffold/Matrix Attachment Region (SAR/MAR binding proteins. Identification and characterization of proteins involved in establishing or maintaining chromosome-scaffold interactions is necessary to understand how the nucleus is organized and how dynamic changes in attachment are correlated with alterations in gene expression. We identified and characterized one such scaffold attachment factor, a Drosophila homolog of mammalian SAF-B. The large nuclei and chromosomes of Drosophila have allowed us to show that SAF-B inhabits distinct subnuclear compartments, forms weblike continua in nuclei of salivary glands, and interacts with discrete chromosomal loci in interphase nuclei. These interactions appear mediated either by DNA-protein interactions, or through RNA-protein interactions that can be altered during changes in gene expression programs. Extraction of soluble nuclear proteins and DNA leaves SAF-B intact, showing that this scaffold/matrix-attachment protein is a durable component of the nuclear matrix. Together, we have shown that SAF-B links the nuclear scaffold, chromosomes, and transcriptional activity.

  2. Low ozone concentrations stimulate cytoskeletal organization, mitochondrial activity and nuclear transcription

    Directory of Open Access Journals (Sweden)

    M. Costanzo

    2015-04-01

    Full Text Available Ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone concentrations and used in medicine as an alternative/adjuvant treatment for different diseases. However, the cellular mechanisms accounting for the positive effects of mild ozonization are still largely unexplored. To this aim, in the present study the effects of low ozone concentrations (1 to 20 µg O3/mL O2 on structural and functional cell features have been investigated in vitro by using morphological, morphometrical, cytochemical and immunocytochemical techniques at bright field, fluorescence and transmission electron microscopy. Cells exposed to pure O2 or air served as controls. The results demonstrated that the effects of ozoneadministration are dependent on gas concentration, and the cytoskeletal organization, mitochondrial activity and nuclear transcription may be differently affected. This suggests that, to ensure effective and permanent metabolic cell activation, ozone treatments should take into account the cytological and cytokinetic features of the different tissues. 

  3. Regulation of transcription through light-activation and light-deactivation of triplex-forming oligonucleotides in mammalian cells.

    Science.gov (United States)

    Govan, Jeane M; Uprety, Rajendra; Hemphill, James; Lively, Mark O; Deiters, Alexander

    2012-07-20

    Triplex-forming oligonucleotides (TFOs) are efficient tools to regulate gene expression through the inhibition of transcription. Here, nucleobase-caging technology was applied to the temporal regulation of transcription through light-activated TFOs. Through site-specific incorporation of caged thymidine nucleotides, the TFO:DNA triplex formation is blocked, rendering the TFO inactive. However, after a brief UV irradiation, the caging groups are removed, activating the TFO and leading to the inhibition of transcription. Furthermore, the synthesis and site-specific incorporation of caged deoxycytidine nucleotides within TFO inhibitor sequences was developed, allowing for the light-deactivation of TFO function and thus photochemical activation of gene expression. After UV-induced removal of the caging groups, the TFO forms a DNA dumbbell structure, rendering it inactive, releasing it from the DNA, and activating transcription. These are the first examples of light-regulated TFOs and their application in the photochemical activation and deactivation of gene expression. In addition, hairpin loop structures were found to significantly increase the efficacy of phosphodiester DNA-based TFOs in tissue culture. PMID:22540192

  4. Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Zhao, Shaomin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Song, Langying [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Manyuan [School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Jiao, Kai, E-mail: kjiao@uab.edu [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2013-11-29

    Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, the activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.

  5. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta

    2013-12-10

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568ASTAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. © Copyright 2014 Federation of European Biochemical Societies. All rights reserved.

  6. Correlation of methane production and functional gene transcriptional activity in a peat soil.

    Science.gov (United States)

    Freitag, Thomas E; Prosser, James I

    2009-11-01

    The transcription dynamics of subunit A of the key gene in methanogenesis (methyl coenzyme M reductase; mcrA) was studied to evaluate the relationship between process rate (methanogenesis) and gene transcription dynamics in a peat soil ecosystem. Soil methanogen process rates were determined during incubation of peat slurries at temperatures from 4 to 37 degrees C, and real-time quantitative PCR was applied to quantify the abundances of mcrA genes and transcripts; corresponding transcriptional dynamics were calculated from mcrA transcript/gene ratios. Internal standards suggested unbiased recovery of mRNA abundances in comparison to DNA levels. In comparison to those in pure-culture studies, mcrA transcript/gene ratios indicated underestimation by 1 order of magnitude, possibly due to high proportions of inactive or dead methanogens. Methane production rates were temperature dependent, with maxima at 25 degrees C, but changes in abundance and transcription of the mcrA gene showed no correlation with temperature. However, mcrA transcript/gene ratios correlated weakly (regression coefficient = 0.76) with rates of methanogenesis. Methanogen process rates increased over 3 orders of magnitude, while the corresponding maximum transcript/gene ratio increase was only 18-fold. mcrA transcript dynamics suggested steady-state expression in peat soil after incubation for 24 and 48 h, similar to that in stationary-phase cultures. mcrA transcript/gene ratios are therefore potential in situ indicators of methanogen process rate changes in complex soil systems.

  7. LIM homeobox transcription factor Lhx2 inhibits skeletal muscle differentiation in part via transcriptional activation of Msx1 and Msx2.

    Science.gov (United States)

    Kodaka, Yusaku; Tanaka, Kiyoko; Kitajima, Kenji; Tanegashima, Kosuke; Matsuda, Ryoichi; Hara, Takahiko

    2015-02-15

    LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.

  8. GCN5 is essential for IRF-4 gene expression followed by transcriptional activation of Blimp-1 in immature B cells.

    Science.gov (United States)

    Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo

    2014-03-01

    During B-cell differentiation, the gene expression of B-cell differentiation-related transcription factors must be strictly controlled by epigenetic mechanisms including histone acetylation and deacetylation, to complete the differentiation pathway. GCN5, one of the most important histone acetyltransferases, is involved in epigenetic events for transcriptional regulation through alterations in the chromatin structure. In this study, by analyzing the homozygous DT40 mutants GCN5(-/-), generated with gene targeting techniques, we found that GCN5 was necessary for transcriptional activation of IRF-4, an essential transcription factor for plasma cell differentiation. GCN5 deficiency caused drastic decreases in both the mRNA and the protein levels of Blimp-1 and IRF-4. The ectopic expression of Blimp-1 and IRF-4 suggests that IRF-4, but not Blimp-1, is the target gene of GCN5 in immature B cells. Moreover, a chromatin immunoprecipitation assay showed that GCN5 bound to the IRF-4 gene around its 5'-flanking region and acetylated H3K9 residues within chromatin surrounding the region in vivo, suggesting that gene expression of IRF-4 is certainly regulated by GCN5. These results reveal that GCN5 is essential for IRF-4 gene expression, followed by transcriptional activation of Blimp-1, and plays a key role in epigenetic regulation of B-cell differentiation.

  9. AhR transcriptional activity in serum of Inuits across Greenlandic districts

    Directory of Open Access Journals (Sweden)

    Bonefeld-Jorgensen Eva C

    2007-10-01

    Full Text Available Abstract Background Human exposure to lipophilic persistent organic pollutants (POPs including polychlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs, polychlorinated biphenyls (PCBs and organochlorine pesticide is ubiquitous. The individual is exposed to a complex mixture of POPs being life-long beginning during critical developmental windows. Exposure to POPs elicits a number of species- and tissue-specific toxic responses, many of which involve the aryl hydrocarbon receptor (AhR. The aim of this study was to compare the actual level of integrated AhR transcriptional activity in the lipophilic serum fraction containing the actual POP mixture among Inuits from different districts in Greenland, and to evaluate whether the AhR transactivity is correlated to the bio-accumulated POPs and/or lifestyle factors. Methods The study included 357 serum samples from the Greenlandic districts: Nuuk and Sisimiut (South West Coast, Qaanaaq (North Coast and Tasiilaq (East Coast. The bio-accumulated serum POPs were extracted by ethanol: hexane and clean-up on Florisil columns. Effects of the serum extract on the AhR transactivity was determined using the Hepa 1.12cR mouse hepatoma cell line carrying an AhR-luciferase reporter gene, and the data was evaluated for possible association to the serum levels of 14 PCB congeners, 10 organochlorine pesticide residues and/or lifestyle factors. Results In total 85% of the Inuit samples elicited agonistic AhR transactivity in a district dependent pattern. The median level of the AhR-TCDD equivalent (AhR-TEQ of the separate genders was similar in the different districts. For the combined data the order of the median AhR-TEQ was Tasiilaq > Nuuk ≥ Sisimiut > Qaanaaq possibly being related to the different composition of POPs. In overall, the AhR transactivity was inversely correlated to the levels of sum POPs, age and/or intake of marine food. Conclusion i We observed that the proportion of dioxin like (DL compounds in the

  10. Signal transducer and activator of transcription 5 is implicated in disease activity in adult and juvenile onset systemic lupus erythematosus.

    Science.gov (United States)

    Meshaal, Safa; El Refai, Rasha; El Saie, Ahmed; El Hawary, Rabab

    2016-06-01

    The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is one of a handful of pleiotropic cascades used to transduce a multitude of signals for development and homeostasis in humans. It is the principal signaling mechanism for a wide array of cytokines and growth factors. Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE). In this study, we tried to assess the role of STAT5 in systemic lupus erythematosus and correlate its phosphorylation level with the disease activity. The activation of the STAT5 was assessed by measuring the level of expression of phosphorylated STAT5 (pSTAT5) using flow cytometry on the peripheral blood T and B cells in 58 SLE patients (40 adult and 18 juvenile onset) and on 23 healthy age- and sex-matched controls for both groups. Serum prolactin level was also assessed in the patients and control by ELISA. The study revealed that the level of pSTAT5 was higher in adult SLE patients than in healthy control (p = 0.001) and in juvenile-onset SLE patients versus age-matched control (p = 0.031). A positive correlation existed between the pSTAT5 levels and Systemic Lupus Activity Measure (SLAM) score and also with multiple clinical manifestations indicating a potential role of STAT5 signaling in pathogenesis SLE. The pSTAT5 signaling is implicated in the disease activity of SLE and may be a useful target of therapy by correcting the dysregulation of cytokines involved in the disease pathogenesis. PMID:27041383

  11. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    Energy Technology Data Exchange (ETDEWEB)

    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan); Oritani, Kenji [Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Matsuda, Tadashi, E-mail: tmatsuda@pharm.hokudai.ac.jp [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan)

    2015-07-31

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR.

  12. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    International Nuclear Information System (INIS)

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR

  13. DcE2F, a functional plant E2F-like transcriptional activator from Daucus carota

    DEFF Research Database (Denmark)

    Albani, D; Mariconti, L; Ricagno, S;

    2000-01-01

    In animal cells the progression of the cell cycle through G(1)/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis...

  14. Effects of antiandrogens on transformation and transcription activation of wild-type and mutated (LNCaP) androgen receptors

    NARCIS (Netherlands)

    C.A. Berrevoets (Cor); J. Veldscholte (Jos); E. Mulder (Eppo)

    1993-01-01

    textabstractLNCaP cells contain androgen receptors with a mutation in the steroid binding domain (Thr 868 changed to Ala) resulting in a changed hormone specificity. Both the wild-type and mutated androgen receptors were transfected into COS cells. Transcription activation was studied in cells co-tr

  15. Aldehyde dehydrogenase 1A1 stabilizes transcription factor Gli2 and enhances the activity of Hedgehog signaling in hepatocellular cancer.

    Science.gov (United States)

    Yan, Zhengwei; Xu, Liyao; Zhang, Junyan; Lu, Quqin; Luo, Shiwen; Xu, Linlin

    2016-03-18

    The Gli transcription factors are primary transcriptional regulators that mediate the activation of Hedgehog (Hh) signaling. Recent studies have revealed that Gli proteins are also regulated transcriptionally and post-translationally through noncanonical mechanisms, independent of Hh signaling. However, the precise mechanisms involved in the regulation of Gli proteins remain unclear. Using a differential mass-spectrometry approach, we found that aldehyde dehydrogenase 1A1 (ALDH1A1) is associated with transcription factor Gli2. Overexpression of ALDH1A1 increased Gli2 protein levels; in contrast, ALDH1A1 depletion facilitated Gli2 degradation. In addition, Gli2 mRNA expression was not affected by ectopic expression of ALDH1A1, indicating the role of ALDH1A1 in the stabilization of Gli2. Further investigation showed that ALDH1A1 prolonged the stability of Gli2 protein in a catalytic-independent manner. Finally, we showed that overexpression of ALDH1A1 activated the Hh signaling pathway and promoted cell growth, migration and invasion in hepatocellular cancer cells. Together, these results illustrate regulatory roles of ALDH1A1 in the activation of the Hh signaling pathway and highlight a novel mechanism for the aberrant activation of the Hh signaling pathway in hepatocellular cancer cells. PMID:26896768

  16. Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1*S⃞

    OpenAIRE

    Choi, Won-Il; Jeon, Bu-Nam; Yun, Chae-Ok; Kim, Pyung-Hwan; Kim, Sung-Eun; Choi, Kang-Yell; Kim, Se Hoon; Hur, Man-Wook

    2009-01-01

    Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptiona...

  17. Proto-oncogene FBI-1 (Pokemon) and SREBP-1 synergistically activate transcription of fatty-acid synthase gene (FASN).

    Science.gov (United States)

    Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F; Hur, Man-Wook

    2008-10-24

    FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

  18. Effects of chronic renal failure rat serum on histone acetyltransferase p300 and activation of activating transcription factor 4 of arterial smooth muscle cells cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    张耀全

    2014-01-01

    Objective To investigate the effects of the rat serum with chronic renal failure(CRF)on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4)of rat arterial vascular smooth muscle cells(VSMCs)cultured in vitro,and explore the possible mechanism.Methods Objective To establish the rat model of

  19. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene

    DEFF Research Database (Denmark)

    Soravia, E; Grebe, A; De Luca, P;

    1995-01-01

    have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes...... and contains relatively GC-rich proximal sequences. A subregion of this sequence, highly conserved between human and murine genes, contains most of the promoter activity and is specifically bound by HeLa nuclear proteins, one of which belongs to the SP1 class....

  20. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    GUO HongXiong; CUN Wei; LIU LongDing; WANG LiChun; ZHAO HongLing; DONG ChengHong; LI QiHan

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular transcriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells' entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  1. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular tran-scriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells’ entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  2. The dimerization domain of SOX9 is required for transcription activation of a chondrocyte-specific chromatin DNA template

    OpenAIRE

    Coustry, Françoise; Oh, Chun-do; Hattori, Takako; Maity, Sankar N.; de Crombrugghe, Benoit; Yasuda, Hideyo

    2010-01-01

    Mutations in SOX9, a gene essential for chondrocyte differentiation cause the human disease campomelic dysplasia (CD). To understand how SOX9 activates transcription, we characterized the DNA binding and cell-free transcription ability of wild-type SOX9 and a dimerization domain SOX9 mutant. Whereas formation of monomeric mutant SOX9–DNA complex increased linearly with increasing SOX9 concentrations, formation of a wild-type SOX9–DNA dimeric complex increased more slowly suggesting a more sig...

  3. Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia

    DEFF Research Database (Denmark)

    Müller-Tidow, Carsten; Klein, Hans-Ulrich; Hascher, Antje;

    2010-01-01

    . The altered genomic regions showed an overrepresentation of cis-binding sites for ets and c-AMP response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML......Acute Myeloid Leukemia (AML) is commonly associated with alterations in transcription factors due to altered expression or gene mutations. These changes might induce leukemia- specific patterns of histone modifications. We used ChIP-Chip to analyze histone H3 Lysine 9 trimethylation (H3K9me3...

  4. Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1.

    Science.gov (United States)

    Guja, Kip E; Venkataraman, Krithika; Yakubovskaya, Elena; Shi, Hui; Mejia, Edison; Hambardjieva, Elena; Karzai, A Wali; Garcia-Diaz, Miguel

    2013-09-01

    Eukaryotic transcription factor B (TFB) proteins are homologous to KsgA/Dim1 ribosomal RNA (rRNA) methyltransferases. The mammalian TFB1, mitochondrial (TFB1M) factor is an essential protein necessary for mitochondrial gene expression. TFB1M mediates an rRNA modification in the small ribosomal subunit and thus plays a role analogous to KsgA/Dim1 proteins. This modification has been linked to mitochondrial dysfunctions leading to maternally inherited deafness, aminoglycoside sensitivity and diabetes. Here, we present the first structural characterization of the mammalian TFB1 factor. We have solved two X-ray crystallographic structures of TFB1M with (2.1 Å) and without (2.0 Å) its cofactor S-adenosyl-L-methionine. These structures reveal that TFB1M shares a conserved methyltransferase core with other KsgA/Dim1 methyltransferases and shed light on the structural basis of S-adenosyl-L-methionine binding and methyltransferase activity. Together with mutagenesis studies, these data suggest a model for substrate binding and provide insight into the mechanism of methyl transfer, clarifying the role of this factor in an essential process for mitochondrial function. PMI