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Sample records for active transcriptional repressor

  1. A TATA sequence-dependent transcriptional repressor activity associated with mammalian transcription factor IIA.

    Aso, T.; Serizawa, H; Conaway, R C; Conaway, J W

    1994-01-01

    In the process of characterizing cellular proteins that modulate basal transcription by RNA polymerase II, we identified a novel repressor activity specific for promoters containing consensus TATA boxes. This activity strongly represses TATA-binding protein (TBP)-dependent transcription initiation from core promoter elements containing a consensus TATA sequence, but activates TBP-dependent transcription from core promoter elements lacking a consensus TATA sequence. Purification of this activi...

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

    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.

  3. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

    Xingguo Zhu

    Full Text Available The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

  4. The Groucho co-repressor is primarily recruited to local target sites in active chromatin to attenuate transcription.

    Aamna Kaul

    2014-08-01

    Full Text Available Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling, and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase. We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in "active" chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone

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

    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

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

    Hjortoe, G.M.; Weilguny, D.; Willumsen, Berthe Marie

    2005-01-01

    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...

  7. Arabidopsis WUSCHEL is a bifunctional transcription factor that acts as a repressor in stem cell regulation and as an activator in floral patterning.

    Ikeda, Miho; Mitsuda, Nobutaka; Ohme-Takagi, Masaru

    2009-11-01

    Most transcription factors act either as activators or repressors, and no such factors with dual function have been unequivocally identified and characterized in plants. We demonstrate here that the Arabidopsis thaliana protein WUSCHEL (WUS), which regulates the maintenance of stem cell populations in shoot meristems, is a bifunctional transcription factor that acts mainly as a repressor but becomes an activator when involved in the regulation of the AGAMOUS (AG) gene. We show that the WUS box, which is conserved among WOX genes, is the domain that is essential for all the activities of WUS, namely, for regulation of stem cell identity and size of floral meristem. All the known activities of WUS were eliminated by mutation of the WUS box, including the ability of WUS to induce the expression of AG. The mutation of the WUS box was complemented by fusion of an exogenous repression domain, with resultant induction of somatic embryogenesis in roots and expansion of floral meristems as observed upon ectopic expression of WUS. By contrast, fusion of an exogenous activation domain did not result in expanded floral meristems but induced flowers similar to those induced by the ectopic expression of AG. Our results demonstrate that WUS acts mainly as a repressor and that its function changes from that of a repressor to that of an activator in the case of regulation of the expression of AG. PMID:19897670

  8. H-NS is a repressor of the Proteus mirabilis urease transcriptional activator gene ureR.

    Coker, C; Bakare, O O; Mobley, H L

    2000-05-01

    Expression of Proteus mirabilis urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A(6)TA(2)CA(2)TGGTA(5)GA(6)TGA(5), is located 16 bp upstream of the sigma(70)-like ureR promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured beta-galactosidase activity of wild-type Escherichia coli MC4100 (H-NS(+)) and its isogenic derivative ATM121 (hns::Tn10) (H-NS(-)) harboring a ureR-lacZ operon fusion plasmid (pLC9801). beta-Galactosidase activity in the H-NS(-) host strain was constitutive and sevenfold greater (P mirabilis hns was able to complement and restore repression of the ureR promoter in the H-NS(-) host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in beta-galactosidase activity in the H-NS(+) host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS(-) host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of P. mirabilis) was equivalent in both the H-NS(-) background and the H-NS(+) background in the presence of urea but was eightfold greater (P = 0.0001) in the H-NS(-) background in the absence of urea. We conclude that H-NS represses ureR expression in the absence of urea induction. PMID:10762273

  9. The transcriptional repressor domain of Gli3 is intrinsically disordered.

    Robert Tsanev

    Full Text Available The transcription factor Gli3 is acting mainly as a transcriptional repressor in the Sonic hedgehog signal transduction pathway. Gli3 contains a repressor domain in its N-terminus from residue G106 to E236. In this study we have characterized the intracellular structure of the Gli3 repressor domain using a combined bioinformatics and experimental approach. According to our findings the Gli3 repressor domain while being intrinsically disordered contains predicted anchor sites for partner interactions. The obvious interaction partners to test were Ski and DNA; however, with both of these the structure of Gli3 repressor domain remained disordered. To locate residues important for the repressor function we mutated several residues within the Gli3 repressor domain. Two of these, H141A and H157N, targeting predicted helical regions, significantly decreased transcriptional repression and thus identify important functional parts of the domain.

  10. The Transcriptional Repressor DEC2 Regulates Sleep Length in Mammals

    Ying HE; Christopher R. Jones; Fujiki, Nobuhiro; Xu, Ying; Guo, Bin; Holder, Jimmy L.; Rossner, Moritz J.; Nishino, Seiji; Fu, Ying-Hui

    2009-01-01

    Sleep deprivation can impair human health and performance. Habitual total sleep time and homeostatic sleep response to sleep deprivation are quantitative traits in humans. Genetic loci for these traits have been identified in model organisms, but none of these potential animal models have a corresponding human genotype and phenotype. We have identified a mutation in a transcriptional repressor (hDEC2-P385R) that is associated with a human short sleep phenotype. Activity profiles and sleep rec...

  11. The B-subdomain of the Xenopus laevis XFIN KRAB-AB domain is responsible for its weaker transcriptional repressor activity compared to human ZNF10/Kox1.

    Nadine Born

    Full Text Available The Krüppel-associated box (KRAB domain interacts with the nuclear hub protein TRIM28 to initiate or mediate chromatin-dependent processes like transcriptional repression, imprinting or suppression of endogenous retroviruses. The prototype KRAB domain initially identified in ZNF10/KOX1 encompasses two subdomains A and B that are found in hundreds of zinc finger transcription factors studied in human and murine genomes. Here we demonstrate for the first time transcriptional repressor activity of an amphibian KRAB domain. After sequence correction, the updated KRAB-AB domain of zinc finger protein XFIN from the frog Xenopus laevis was found to confer transcriptional repression in reporter assays in Xenopus laevis A6 kidney cells as well as in human HeLa, but not in the minnow Pimephales promelas fish cell line EPC. Binding of the XFIN KRAB-AB domain to human TRIM28 was demonstrated in a classical co-immunoprecipitation approach and visualized in a single-cell compartmentalization assay. XFIN-AB displayed reduced potency in repression as well as lower strength of interaction with TRIM28 compared to ZNF10 KRAB-AB. KRAB-B subdomain swapping between the two KRAB domains indicated that it was mainly the KRAB-B subdomain of XFIN that was responsible for its lower capacity in repression and binding to human TRIM28. In EPC fish cells, ZNF10 and XFIN KRAB repressor activity could be partially restored to low levels by adding exogenous human TRIM28. In contrast to XFIN, we did not find any transcriptional repression activity for the KRAB-like domain of human PRDM9 in HeLa cells. PRDM9 is thought to harbor an evolutionary older domain related to KRAB whose homologs even occur in invertebrates. Our results support the notion that functional bona fide KRAB domains which confer transcriptional repression and interact with TRIM28 most likely co-evolved together with TRIM28 at the beginning of tetrapode evolution.

  12. The bacterial effector HopX1 targets JAZ transcriptional repressors to activate jasmonate signaling and promote infection in Arabidopsis.

    Selena Gimenez-Ibanez

    2014-02-01

    Full Text Available Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR, which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile. Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.

  13. Gfi1, a transcriptional repressor, inhibits the induction of the T helper type 1 programme in activated CD4 T cells.

    Suzuki, Junpei; Maruyama, Saho; Tamauchi, Hidekazu; Kuwahara, Makoto; Horiuchi, Mika; Mizuki, Masumi; Ochi, Mizuki; Sawasaki, Tatsuya; Zhu, Jinfang; Yasukawa, Masaki; Yamashita, Masakatsu

    2016-04-01

    A transcriptional repressor Gfi1 promotes T helper type 2 (Th2) cell development and inhibits Th17 and inducible regulatory T-cell differentiation. However, the role of Gfi1 in regulating Th1 cell differentiation and the Th1-type immune response remains to be investigated. We herein demonstrate that Gfi1 inhibits the induction of the Th1 programme in activated CD4 T cells. The activated Gfi1-deficient CD4 T cells spontaneously develop into Th1 cells in an interleukin-12- and interferon-γ-independent manner. The increase of Th1-type immune responses was confirmed in vivo in Gfi1-deficient mice using a murine model of nickel allergy and delayed-type hypersensitivity (DTH). The expression levels of Th1-related transcription factors were found to increase in Gfi1-deficient activated CD4 T cells. Tbx21, Eomes and Runx2 were identified as possible direct targets of Gfi1. Gfi1 binds to the Tbx21, Eomes and Runx2 gene loci and reduces the histone H3K4 methylation levels in part by modulating Lsd1 recruitment. Together, these findings demonstrate a novel regulatory role of Gfi1 in the regulation of the Th1-type immune response. PMID:26749286

  14. A novel GDNF-inducible gene, BMZF3, encodes a transcriptional repressor associated with KAP-1

    The Krueppel-associated box (KRAB)-containing zinc finger proteins (ZFPs) comprise the largest family of zinc finger transcription factors that function as transcriptional repressors. In the study of glial cell line-derived neurotrophic factor (GDNF)-RET signaling, we have identified bone marrow zinc finger 3 (BMZF3), encoding a KRAB-ZFP, as a GDNF-inducible gene by differential display analysis. The expression of BMZF3 transcripts in the human neuroblastoma cell line TGW increased 1 h after GDNF stimulation, as determined by Northern blotting and quantitative reverse-transcriptase polymerase chain reaction. The BMZF3 possesses transcriptional repressor activity in the KRAB domain. BMZF3 interacts with a co-repressor protein, KRAB-associated protein 1 (KAP-1), through the KRAB domain and siRNA-mediated knockdown of KAP-1 abolished the transcriptional repressor activity of BMZF3, indicating that KAP-1 is necessary for BMZF3 function. Furthermore, siRNA-mediated silencing of BMZF3 inhibited cell proliferation. These findings suggest that BMZF3 is a transcriptional repressor induced by GDNF that plays a role in cell proliferation

  15. Down-regulation of the zinc-finger homeobox protein TSHZ2 releases GLI1 from the nuclear repressor complex to restore its transcriptional activity during mammary tumorigenesis.

    Riku, Miho; Inaguma, Shingo; Ito, Hideaki; Tsunoda, Takumi; Ikeda, Hiroshi; Kasai, Kenji

    2016-02-01

    Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1. PMID:26744317

  16. Glutamate activates c-fos in glial cells via a novel mechanism involving the glutamate receptor subtype mGlu5 and the transcriptional repressor DREAM.

    Edling, Ylva; Ingelman-Sundberg, Magnus; Simi, Anastasia

    2007-02-01

    Activation of c-fos in brain is related to coupling of neuronal activity to gene expression, but also to pathological conditions such as seizures or excitotoxicity-induced cell death. Glutamate activates c-fos in neurons through the calcium-dependent phosphorylation of CREB by ERK and/or CaMKIV kinase pathways downstream NMDA-receptors. In glial cells, however, the activation of c-fos by glutamate is poorly understood. Because glial cells actively modulate neuronal excitability and the brain's response to injury, we studied the mechanisms by which glutamate activates c-fos in rat cortical glial cells. Glutamate potently induced c-fos mRNA in a calcium-dependent manner, as demonstrated by using the calcium chelator BAPTA-AM. Glutamate-induced c-fos mRNA expression was not sensitive to inhibitors of ERK, p38(MAPK), or CaMK pathways, indicating that glial c-fos is activated by a distinct mechanism. Thapsigargin abolished the glutamate effect on c-fos mRNA, indicating ER calcium mobilization. Additionally, glutamate induction of c-fos mRNA was sensitive to the mGluR5 antagonist MPEP but not the NMDA-R antagonist MK-801. In luciferase reporter assays, DRE, which actively represses c-fos by binding the calcium-binding transcriptional repressor DREAM, was activated by glutamate, whereas SRE and CRE were not. Finally, glutamate caused the nuclear export of DREAM in astrocytes, and transfection of astrocytes with a mutant variant of DREAM that constitutively binds DNA inhibited glutamate-induced c-Fos expression. These findings are in sharp contrast to the mechanism described in neurons and suggest a novel pathway activated by glutamate in glial cells that employs mGluR5, ER calcium, and the derepression of c-fos at the DRE. PMID:17120244

  17. The E2 transcriptional repressor can compensate for Sp1 activation of the human papillomavirus type 18 early promoter.

    Demeret, C; Yaniv, M; Thierry, F

    1994-01-01

    The E6/E7 early promoter (P105) of genital human papillomavirus type 18 contains binding sites for the viral regulator E2, tandemly repeated and closely flanked by two crucial promoter elements; the TATA box downstream and an Sp1 binding site upstream. We showed that binding of purified E2 and Sp1 proteins in vitro to their neighboring sites is mutually exclusive and that Sp1 is displaced by E2. However, this displacement did not result in repression of P105 transcription. In contrast, bindin...

  18. Regulation of phage Mu repressor transcription by IHF depends on the level of the early transcription.

    van Rijn, P A; Goosen, N; Turk, S C; van de Putte, P

    1989-01-01

    Integration Host Factor (IHF) of E. coli can stimulate both early and repressor transcription of bacteriophage Mu. We introduced several mutations in the early promoter (Pe) and studied the effect of these mutations on the stimulation of early and repressor transcription by IHF. All mutant promoters are still positive regulated by IHF, but the level of stimulation is dependent on the strength of the promoter. The strength of the early promoter has an even greater impact on the regulation of t...

  19. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    Mahfouz, Magdy M.

    2011-12-14

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  20. Changing a conserved amino acid in R2R3-MYB transcription repressors results in cytoplasmic accumulation and abolishes their repressive activity in Arabidopsis.

    Zhou, Meiliang; Sun, Zhanmin; Wang, Chenglong; Zhang, Xinquan; Tang, Yixiong; Zhu, Xuemei; Shao, Jirong; Wu, Yanmin

    2015-10-01

    Sub-group 4 R2R3-type MYB transcription factors, including MYB3, MYB4, MYB7 and MYB32, act as repressors in phenylpropanoid metabolism. These proteins contain the conserved MYB domain and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) repression domain. Additionally, MYB4, MYB7 and MYB32 possess a putative zinc-finger domain and a conserved GY/FDFLGL motif in their C-termini. The protein 'sensitive to ABA and drought 2' (SAD2) recognizes the nuclear pore complex, which then transports the SAD2-MYB4 complex into the nucleus. Here, we show that the conserved GY/FDFLGL motif contributes to the interaction between MYB factors and SAD2. The Asp → Asn mutation in the GY/FDFLGL motif abolishes the interaction between MYB transcription factors and SAD2, and therefore they cannot be transported into the nucleus and cannot repress their target genes. We found that MYB4(D261N) loses the capacity to repress expression of the cinnamate 4-hydroxylase (C4H) gene and biosynthesis of sinapoyl malate. Our results indicate conservation among MYB transcription factors in terms of their interaction with SAD2. Therefore, the Asp → Asn mutation may be used to engineer transcription factors. PMID:26332741

  1. The intracellular domain of teneurin-1 induces the activity of microphthalmia-associated transcription factor (MITF) by binding to transcriptional repressor HINT1.

    Schöler, Jonas; Ferralli, Jacqueline; Thiry, Stéphane; Chiquet-Ehrismann, Ruth

    2015-03-27

    Teneurins are large type II transmembrane proteins that are necessary for the normal development of the CNS. Although many studies highlight the significance of teneurins, especially during development, there is only limited information known about the molecular mechanisms of function. Previous studies have shown that the N-terminal intracellular domain (ICD) of teneurins can be cleaved at the membrane and subsequently translocates to the nucleus, where it can influence gene transcription. Because teneurin ICDs do not contain any intrinsic DNA binding sequences, interaction partners are required to affect transcription. Here, we identified histidine triad nucleotide binding protein 1 (HINT1) as a human teneurin-1 ICD interaction partner in a yeast two-hybrid screen. This interaction was confirmed in human cells, where HINT1 is known to inhibit the transcription of target genes by directly binding to transcription factors at the promoter. In a whole transcriptome analysis of BS149 glioblastoma cells overexpressing the teneurin-1 ICD, several microphthalmia-associated transcription factor (MITF) target genes were found to be up-regulated. Directly comparing the transcriptomes of MITF versus TEN1-ICD-overexpressing BS149 cells revealed 42 co-regulated genes, including glycoprotein non-metastatic b (GPNMB). Using real-time quantitative PCR to detect endogenous GPNMB expression upon overexpression of MITF and HINT1 as well as promoter reporter assays using GPNMB promoter constructs, we could demonstrate that the teneurin-1 ICD binds HINT1, thus switching on MITF-dependent transcription of GPNMB. PMID:25648896

  2. Inhibition of Yin Yang 1-Dependent Repressor Activity of DR5 Transcription and Expression by the Novel Proteasome Inhibitor NPI-0052 Contributes to its TRAIL-Enhanced Apoptosis in Cancer Cells1

    Baritaki, Stavroula; Suzuki, Eriko; Umezawa, Kazuo; Spandidos, Demetrios A.; Berenson, James; DANIELS, TRACY R.; Penichet, Manuel L; Jazirehi, Ali R; Palladino, Michael; Bonavida, Benjamin

    2008-01-01

    TRAIL promotes apoptotic tumor cell death; however, TRAIL-resistant tumors need to be sensitized to reverse resistance. Proteasome inhibitors potentiate TRAIL apoptosis in vitro and in vivo and correlate with up-regulation of death receptor 5 (DR5) via an unknown mechanism. We hypothesized that the proteasome inhibitor NPI-0052 inhibits the transcription repressor Yin Yang 1 (YY1) which regulates TRAIL resistance and negatively regulates DR5 transcription. Treatment of PC-3 and Ramos cells wi...

  3. Dynamical analysis on gene activity in the presence of repressors and an interfering promoter.

    Nakanishi, Hiizu; Mitarai, Namiko; Sneppen, Kim

    2008-11-01

    Transcription is regulated through interplay among transcription factors, an RNA polymerase (RNAP), and a promoter. Even for a simple repressive transcription factor that disturbs promoter activity at initial binding of RNAP, its repression level is not determined solely by the dissociation constant of transcription factor but is sensitive to timescales of processes in RNAP. We first analyze the promoter activity under strong repression by a slow binding repressor, in which case transcription events occur in bursts, followed by long quiescent periods while a repressor binds to the operator; the number of transcription events, bursting, and quiescent times are estimated by reaction rates. We then examine interference effect from an opposing promoter, using the correlation function of initiation events for a single promoter. The interference is shown to de-repress the promoter because RNAPs from the opposing promoter most likely encounter the repressor and remove it in case of strong repression. This de-repression mechanism should be especially prominent for the promoters that facilitate fast formation of open complex with the repressor whose binding rate is slower than approximately 1/s. Finally, we discuss possibility of this mechanism for high activity of promoter PR in the hyp-mutant of lambda-phage. PMID:18658208

  4. Plastic downregulation of the transcriptional repressor BCL6 during maturation of human dendritic cells

    Dendritic cell (DC) maturation links peripheral events initiated by the encounter with pathogens to the activation and expansion of antigen-specific T lymphocytes in secondary lymphoid organs. Here, we describe an as yet unrecognized modulator of human DC maturation, the transcriptional repressor BCL6. We found that both myeloid and plasmacytoid DCs constitutively express BCL6, which is rapidly downregulated following maturation triggered by selected stimuli. Both in unstimulated and maturing DCs, control of BCL6 protein levels reflects the convergence of several mechanisms regulating BCL6 stability, mRNA transcription and nuclear export. By regulating the induction of several genes implicated in the immune response, including inflammatory cytokines, chemokines and survival genes, BCL6 may represent a pivotal modulator of the afferent branch of the immune response

  5. FILAMENTOUS FLOWER controls lateral organ development by acting as both an activator and a repressor

    Bonaccorso Oliver

    2012-10-01

    Full Text Available Abstract Background The YABBY (YAB family of transcription factors participate in a diverse range of processes that include leaf and floral patterning, organ growth, and the control of shoot apical meristem organisation and activity. How these disparate functions are regulated is not clear, but based on interactions with the LEUNIG-class of co-repressors, it has been proposed that YABs act as transcriptional repressors. In the light of recent work showing that DNA-binding proteins associated with the yeast co-repressor TUP1 can also function as activators, we have examined the transcriptional activity of the YABs. Results Of the four Arabidopsis YABs tested in yeast, only FILAMENTOUS FLOWER (FIL activated reporter gene expression. Similar analysis with Antirrhinum YABs identified the FIL ortholog GRAMINIFOLIA as an activator. Plant-based transactivation assays not only confirmed the potential of FIL to activate transcription, but also extended this property to the FIL paralog YABBY3 (YAB3. Subsequent transcriptomic analysis of lines expressing a steroid-inducible FIL protein revealed groups of genes that responded either positively or negatively to YAB induction. Included in the positively regulated group of genes were the polarity regulators KANADI1 (KAN1, AUXIN RESPONSE FACTOR 4 (ARF4 and ASYMMETRIC LEAVES1 (AS1. We also show that modifying FIL to function as an obligate repressor causes strong yab loss-of-function phenotypes. Conclusions Collectively these data show that FIL functions as a transcriptional activator in plants and that this activity is involved in leaf patterning. Interestingly, our study also supports the idea that FIL can act as a repressor, as transcriptomic analysis identified negatively regulated FIL-response genes. To reconcile these observations, we propose that YABs are bifunctional transcription factors that participate in both positive and negative regulation. These findings fit a model of leaf development in which

  6. The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of “off Target” Gene (glnE Expression

    Paul J. T. Johnson

    2015-06-01

    Full Text Available MtrR is a well-characterized repressor of the Neisseria gonorrhoeae mtrCDE efflux pump operon. However, results from a previous transcriptional profiling study suggested that MtrR also represses or activates expression of at least sixty genes outside of the mtr locus. Evidence that MtrR can directly repress so-called “off target” genes has previously been reported; in particular, MtrR was shown to directly repress glnA, which encodes glutamine synthetase. In contrast, evidence for the ability of MtrR to directly activate expression of gonococcal genes has been lacking; herein, we provide such evidence. We now report that MtrR has the ability to directly activate expression of glnE, which encodes the dual functional adenyltransferase/deadenylase enzyme GlnE that modifies GlnA resulting in regulation of its role in glutamine biosynthesis. With its capacity to repress expression of glnA, the results presented herein emphasize the diverse and often opposing regulatory properties of MtrR that likely contributes to the overall physiology and metabolism of N. gonorrhoeae.

  7. Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

    Christensen, Jesper; Cloos, Paul; Toftegaard, Ulla;

    2005-01-01

    The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family...

  8. The transcriptional repressor DREAM is involved in thyroid gene expression

    Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds to DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca2+ interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function

  9. The histone-like protein H-NS acts as a transcriptional repressor for expression of the anaerobic and growth phase activator AppY of Escherichia coli

    Atlung, Tove; Sund, Susanne; Olesen, Kirsten;

    1996-01-01

    unusually AT-rich DNA. The nucleoid-associated protein H-NS has a DNA-binding specificity for intrinscally curved AT-rich DNA. Using a single-copy transcriptional appY-lacZ fusion, we have shown that appY gene expression is derepressed in hns mutants during aerobic exponential growth. In the hns mutant......, growth phase and growth rate regulation under aerobic conditions was maintained, while ArcA-dependent anaerobic induction was greatly diminished. Judged by two-dimensional gel electrophoresis, the appY promoter fragment exhibits the features characteristic of curved DNA. Gel retardation assays showed...

  10. WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance

    Yokotani, Naoki; Sato, Yuko; Tanabe, Shigeru; Chujo, Tetsuya; Shimizu, Takafumi; Okada, Kazunori; Yamane, Hisakazu; Shimono, Masaki; Sugano, Shoji; Takatsuji, Hiroshi; Kaku, Hisatoshi; Minami, Eiichi; Nishizawa, Yoko

    2013-01-01

    OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice ce...

  11. Change of function of the wheat stress-responsive transcriptional repressor TaRAP2.1L by repressor motif modification.

    Amalraj, Amritha; Luang, Sukanya; Kumar, Manoj Yadav; Sornaraj, Pradeep; Eini, Omid; Kovalchuk, Nataliya; Bazanova, Natalia; Li, Yuan; Yang, Nannan; Eliby, Serik; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy

    2016-02-01

    Plants respond to abiotic stresses by changes in gene regulation, including stress-inducible expression of transcriptional activators and repressors. One of the best characterized families of drought-related transcription factors are dehydration-responsive element binding (DREB) proteins, known as C-repeat binding factors (CBF). The wheat DREB/CBF gene TaRAP2.1L was isolated from drought-affected tissues using a dehydration-responsive element (DRE) as bait in a yeast one-hybrid screen. TaRAP2.1L is induced by elevated abscisic acid, drought and cold. A C-terminal ethylene responsive factor-associated amphiphilic repression (EAR) motif, known to be responsible for active repression of target genes, was identified in the TaRAP2.1L protein. It was found that TaRAP2.1L has a unique selectivity of DNA-binding, which differs from that of DREB activators. This binding selectivity remains unchanged in a TaRAP2.1L variant with an inactivated EAR motif (TaRAP2.1Lmut). To study the role of the TaRAP2.1L repressor activity associated with the EAR motif in planta, transgenic wheat overexpressing native or mutated TaRAP2.1L was generated. Overexpression of TaRAP2.1L under constitutive and stress-inducible promoters in transgenic wheat and barley led to dwarfism and decreased frost tolerance. By contrast, constitutive overexpression of the TaRAP2.1Lmut gene had little or no negative influence on wheat development or grain yield. Transgenic lines with the TaRAP2.1Lmut transgene had an enhanced ability to survive frost and drought. The improved stress tolerance is attributed to up-regulation of several stress-related genes known to be downstream genes of DREB/CBF activators. PMID:26150199

  12. In vitro transcription accurately predicts lac repressor phenotype in vivo in Escherichia coli

    Matthew Almond Sochor

    2014-07-01

    Full Text Available A multitude of studies have looked at the in vivo and in vitro behavior of the lac repressor binding to DNA and effector molecules in order to study transcriptional repression, however these studies are not always reconcilable. Here we use in vitro transcription to directly mimic the in vivo system in order to build a self consistent set of experiments to directly compare in vivo and in vitro genetic repression. A thermodynamic model of the lac repressor binding to operator DNA and effector is used to link DNA occupancy to either normalized in vitro mRNA product or normalized in vivo fluorescence of a regulated gene, YFP. An accurate measurement of repressor, DNA and effector concentrations were made both in vivo and in vitro allowing for direct modeling of the entire thermodynamic equilibrium. In vivo repression profiles are accurately predicted from the given in vitro parameters when molecular crowding is considered. Interestingly, our measured repressor–operator DNA affinity differs significantly from previous in vitro measurements. The literature values are unable to replicate in vivo binding data. We therefore conclude that the repressor-DNA affinity is much weaker than previously thought. This finding would suggest that in vitro techniques that are specifically designed to mimic the in vivo process may be necessary to replicate the native system.

  13. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    Rizvi, Noreen F; Weaver, Jessica D; Cram, Erin J; Lee-Parsons, Carolyn W T

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis. PMID:27467510

  14. Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

    D' Aquino,J.; Tetenbaum-Novatt, J.; White, A.; Berkovitch, F.; Ringe, D.

    2005-01-01

    The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10{sup -7}, binding site 2 (primary) is a low-affinity binding site with a binding constant of 6.3 x 10{sup -4}. These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A, C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.

  15. The Banana Transcriptional Repressor MaDEAR1 Negatively Regulates Cell Wall-Modifying Genes Involved in Fruit Ripening.

    Fan, Zhong-Qi; Kuang, Jian-Fei; Fu, Chang-Chun; Shan, Wei; Han, Yan-Chao; Xiao, Yun-Yi; Ye, Yu-Jie; Lu, Wang-Jin; Lakshmanan, Prakash; Duan, Xue-Wu; Chen, Jian-Ye

    2016-01-01

    Ethylene plays an essential role in many biological processes including fruit ripening via modulation of ethylene signaling pathway. Ethylene Response Factors (ERFs) are key transcription factors (TFs) involved in ethylene perception and are divided into AP2, RAV, ERF, and DREB sub-families. Although a number of studies have implicated the involvement of DREB sub-family genes in stress responses, little is known about their roles in fruit ripening. In this study, we identified a DREB TF with a EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. Expression analysis indicated that MaDEAR1 expression was repressed by ethylene, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. In addition, MaDEAR1 promoter activity was also suppressed in response to ethylene treatment. More importantly, MaDEAR1 directly binds to the DRE/CRT motifs in promoters of several cell wall-modifying genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 associated with fruit softening during ripening and represses their activities. These data suggest that MaDEAR1 acts as a transcriptional repressor of cell wall-modifying genes, and may be negatively involved in ethylene-mediated ripening of banana fruit. Our findings provide new insights into the involvement of DREB TFs in the regulation of fruit ripening. PMID:27462342

  16. The Banana Transcriptional Repressor MaDEAR1 Negatively Regulates Cell Wall-Modifying Genes Involved in Fruit Ripening

    Fan, Zhong-qi; Kuang, Jian-fei; Fu, Chang-chun; Shan, Wei; Han, Yan-chao; Xiao, Yun-yi; Ye, Yu-jie; Lu, Wang-jin; Lakshmanan, Prakash; Duan, Xue-wu; Chen, Jian-ye

    2016-01-01

    Ethylene plays an essential role in many biological processes including fruit ripening via modulation of ethylene signaling pathway. Ethylene Response Factors (ERFs) are key transcription factors (TFs) involved in ethylene perception and are divided into AP2, RAV, ERF, and DREB sub-families. Although a number of studies have implicated the involvement of DREB sub-family genes in stress responses, little is known about their roles in fruit ripening. In this study, we identified a DREB TF with a EAR motif, designated as MaDEAR1, which is a nucleus-localized transcriptional repressor. Expression analysis indicated that MaDEAR1 expression was repressed by ethylene, with reduced levels of histone H3 and H4 acetylation at its regulatory regions during fruit ripening. In addition, MaDEAR1 promoter activity was also suppressed in response to ethylene treatment. More importantly, MaDEAR1 directly binds to the DRE/CRT motifs in promoters of several cell wall-modifying genes including MaEXP1/3, MaPG1, MaXTH10, MaPL3, and MaPME3 associated with fruit softening during ripening and represses their activities. These data suggest that MaDEAR1 acts as a transcriptional repressor of cell wall-modifying genes, and may be negatively involved in ethylene-mediated ripening of banana fruit. Our findings provide new insights into the involvement of DREB TFs in the regulation of fruit ripening. PMID:27462342

  17. Role of transcriptional repressors in transformation by bovine papillomavirus type 1.

    Zemlo, T R; Lohrbach, B; Lambert, P F

    1994-01-01

    Transformation of rodent cells by bovine papillomavirus type 1 (BPV-1) has been shown to require the direct contribution of the viral oncogenes encoded by the E5, E6, and E7 translational open reading frames (ORFs). It is also known that the viral E1 and E2 ORFs contribute indirectly to cellular transformation through their transcriptional modulation of these viral oncogenes. A mutant BPV-1 disrupted in two of the proteins encoded by the E2 ORF, the E2 transcriptional repressors, has a comple...

  18. Arabidopsis Ovate Family Proteins, a Novel Transcriptional Repressor Family, Control Multiple Aspects of Plant Growth and Development

    Wang, Shucai [University of British Columbia, Vancouver; Chang, Ying [Northeast Agricultural University; Guo, Jianjun [Harvard University; Zeng, Qingning [University of British Columbia, Vancouver; Ellis, Brian [University of British Columbia, Vancouver; Chen, Jay [ORNL

    2011-01-01

    BACKGROUND: The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs), a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7), and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1)-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. CONCLUSIONS/SIGNIFICANCE: Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the first overview of a

  19. Arabidopsis ovate family proteins, a novel transcriptional repressor family, control multiple aspects of plant growth and development.

    Shucai Wang

    Full Text Available BACKGROUND: The Arabidopsis genome contains 18 genes that are predicted to encode Ovate Family Proteins (AtOFPs, a protein family characterized by a conserved OVATE domain, an approximately 70-amino acid domain that was originally found in tomato OVATE protein. Among AtOFP family members, AtOFP1 has been shown to suppress cell elongation, in part, by suppressing the expression of AtGA20ox1, AtOFP4 has been shown to regulate secondary cell wall formation by interact with KNOTTED1-LIKE HOMEODOMAIN PROTEIN 7 (KNAT7, and AtOFP5 has been shown to regulate the activity of a BEL1-LIKEHOMEODOMAIN 1(BLH1-KNAT3 complex during early embryo sac development, but little is known about the function of other AtOFPs. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated here that AtOFP proteins could function as effective transcriptional repressors in the Arabidopsis protoplast transient expression system. The analysis of loss-of-function alleles of AtOFPs suggested AtOFP genes may have overlapping function in regulating plant growth and development, because none of the single mutants identified, including T-DNA insertion mutants in AtOFP1, AtOFP4, AtOFP8, AtOFP10, AtOFP15 and AtOFP16, displayed any apparent morphological defects. Further, Atofp1 Atofp4 and Atofp15 Atofp16 double mutants still did not differ significantly from wild-type. On the other hand, plants overexpressing AtOFP genes displayed a number of abnormal phenotypes, which could be categorized into three distinct classes, suggesting that AtOFP genes may also have diverse functions in regulating plant growth and development. Further analysis suggested that AtOFP1 regulates cotyledon development in a postembryonic manner, and global transcript profiling revealed that it suppress the expression of many other genes. CONCLUSIONS/SIGNIFICANCE: Our results showed that AtOFPs function as transcriptional repressors and they regulate multiple aspects of plant growth and development. These results provided the

  20. Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs.

    Choe, J; Vaillancourt, P; Stenlund, A; Botchan, M

    1989-04-01

    Genetic and biochemical evidence has established that the E2 open reading frame (ORF) of bovine papillomavirus type 1 encodes at least two different site-specific DNA-binding proteins, one which activates and the other which represses expression from a viral promoter (P. F. Lambert, B. A. Spalholz, and P. M. Howley, Cell 50:69-78, 1987). We have obtained data which show that a second form of the repressor gene is expressed in transformed cells harboring stable viral plasmids. The structural details of this gene have been discerned by cDNA cloning, by RNase protection, and by primer extension analysis of in vivo RNA. Moreover, data from in vitro transcription experiments support the notion that this form of the E2 repressor is expressed from a novel viral promoter and that a small exon from another ORF is linked to an active repressor domain in E2. Thus, two different forms of the repressor are expressed from different promoters and might be independently regulated either in the cell cycle or in different tissue types. We show by functional in vivo assays utilizing a cDNA vector encoding this gene that the trans-acting factor has in vivo activities similar to those of the known repressor. Our screen of a cDNA library for cDNA clones representing bovine papillomavirus transcripts has also revealed a number of other novel structures defining new donor and acceptor RNA-processing sites. Notably, clones which conceptually can be translated to yield an E7 protein, the viral M gene, and the entire E2 ORF have been characterized. Finally, truncated versions of putative E8 cDNAs were also obtained. PMID:2538655

  1. Activating PER repressor through a DBT-directed phosphorylation switch.

    Saul Kivimäe

    2008-07-01

    Full Text Available Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT, a Drosophila ortholog of human casein kinase I (CKIepsilon/delta. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER. DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The per(S mutation, which is associated with short-period (19-h circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function.

  2. Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1.

    Bauknecht, T; Angel, P; Royer, H D; zur Hausen, H

    1992-01-01

    The human papillomavirus type 18 (HPV-18) promoter contains a TPA responsive element (TRE) which confers TPA responsiveness on a heterologous promoter. In the context of the HPV-18 promoter, however, this AP-1 site is inactive. We have identified a negative regulatory domain in the HPV-18 promoter which represses the constitutive and TPA-induced AP-1 activity. This negative regulatory sequence has been mapped to 44 nucleotides (OL13). We identified this element as a transcriptional silencer based on its ability to interfere with transcriptional initiation. This HPV-18 silencer domain was narrowed down further to 23 nucleotides, the OL13B element, which bears similarity to three other silencer sequences, present in the mouse N-ras gene upstream regulatory region, the mouse albumin gene enhancer and the adeno-associated virus P5 promoter. The transcriptional repressor protein YY1, which negatively regulates the P5 promoter, binds to the HPV-18 silencer with high affinity. Mutation of the YY1 binding site leads to an enhanced activity of the HPV-18 promoter, strongly suggesting that YY1 plays an important role in controlling HPV-18 early gene expression. Images PMID:1330541

  3. The Mannitol Operon Repressor MTIR belongs to a new class of transcription regulators in bacteria.

    Tan, K.; Borovilos, M.; Zhou, M; Horer, S; Clancy, S; Moy, S; Volkart, LL; Sassoon, J; Baumann, U; Joachimiak, A (Biosciences Division); (Univ. of Berne)

    2009-12-25

    Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representing a novel all {alpha}-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.

  4. The MogR Transcriptional Repressor Regulates Nonhierarchal Expression of Flagellar Motility Genes and Virulence in Listeria monocytogenes.

    2006-04-01

    Full Text Available Flagella are surface structures critical for motility and virulence of many bacterial species. In Listeria monocytogenes, MogR tightly represses expression of flagellin (FlaA during extracellular growth at 37 degrees C and during intracellular infection. MogR is also required for full virulence in a murine model of infection. Using in vitro and in vivo infection models, we determined that the severe virulence defect of MogR-negative bacteria is due to overexpression of FlaA. Specifically, overproduction of FlaA in MogR-negative bacteria caused pleiotropic defects in bacterial division (chaining phenotype, intracellular spread, and virulence in mice. DNA binding and microarray analyses revealed that MogR represses transcription of all known flagellar motility genes by binding directly to a minimum of two TTTT-N(5-AAAA recognition sites positioned within promoter regions such that RNA polymerase binding is occluded. Analysis of MogR protein levels demonstrated that modulation of MogR repression activity confers the temperature-specificity to flagellar motility gene expression. Epistasis analysis revealed that MogR repression of transcription is antagonized in a temperature-dependent manner by the DegU response regulator and that DegU further regulates FlaA levels through a posttranscriptional mechanism. These studies provide the first known example to our knowledge of a transcriptional repressor functioning as a master regulator controlling nonhierarchal expression of flagellar motility genes.

  5. Regulation of gene expression through a transcriptional repressor that senses acyl-chain length in membrane phospholipids.

    Hofbauer, Harald F; Schopf, Florian H; Schleifer, Hannes; Knittelfelder, Oskar L; Pieber, Bartholomäus; Rechberger, Gerald N; Wolinski, Heimo; Gaspar, Maria L; Kappe, C Oliver; Stadlmann, Johannes; Mechtler, Karl; Zenz, Alexandra; Lohner, Karl; Tehlivets, Oksana; Henry, Susan A; Kohlwein, Sepp D

    2014-06-23

    Membrane phospholipids typically contain fatty acids (FAs) of 16 and 18 carbon atoms. This particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues. Here, we show that the relative proportion of C16 versus C18 FAs is regulated by the activity of acetyl-CoA carboxylase (Acc1), the first and rate-limiting enzyme of FA de novo synthesis. Acc1 activity is attenuated by AMPK/Snf1-dependent phosphorylation, which is required to maintain an appropriate acyl-chain length distribution. Moreover, we find that the transcriptional repressor Opi1 preferentially binds to C16 over C18 phosphatidic acid (PA) species: thus, C16-chain containing PA sequesters Opi1 more effectively to the ER, enabling AMPK/Snf1 control of PA acyl-chain length to determine the degree of derepression of Opi1 target genes. These findings reveal an unexpected regulatory link between the major energy-sensing kinase, membrane lipid composition, and transcription. PMID:24960695

  6. The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

    Gullberg Urban

    2010-05-01

    Full Text Available Abstract Background The Eight-Twenty-One (ETO nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16 and myeloid translocation Gene-Related protein 1 (MTGR1. By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function. Results A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells. Conclusions We demonstrate that the GATA-1 transcription factor binds and

  7. Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions.

    Sakamoto, Hideki; Maruyama, Kyonoshin; Sakuma, Yoh; Meshi, Tetsuo; Iwabuchi, Masaki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2004-09-01

    ZPT2-related proteins that have two canonical Cys-2/His-2-type zinc-finger motifs in their molecules are members of a family of plant transcription factors. To characterize the role of this type of protein, we analyzed the function of Arabidopsis L. Heynh. genes encoding four different ZPT2-related proteins (AZF1, AZF2, AZF3, and STZ). Gel-shift analysis showed that the AZFs and STZ bind to A(G/C)T repeats within an EP2 sequence, known as a target sequence of some petunia (Petunia hybrida) ZPT2 proteins. Transient expression analysis using synthetic green fluorescent protein fusion genes indicated that the AZFs and STZ are preferentially localized to the nucleus. These four ZPT2-related proteins were shown to act as transcriptional repressors that down-regulate the transactivation activity of other transcription factors. RNA gel-blot analysis showed that expression of AZF2 and STZ was strongly induced by dehydration, high-salt and cold stresses, and abscisic acid treatment. Histochemical analysis of beta-glucuronidase activities driven by the AZF2 or STZ promoters revealed that both genes are induced in leaves rather than roots of rosette plants by the stresses. Transgenic Arabidopsis overexpressing STZ showed growth retardation and tolerance to drought stress. These results suggest that AZF2 and STZ function as transcriptional repressors to increase stress tolerance following growth retardation. PMID:15333755

  8. Control of Enzyme IIscr and Sucrose-6-Phosphate Hydrolase Activities in Streptococcus mutans by Transcriptional Repressor ScrR Binding to the cis-Active Determinants of the scr Regulon

    Wang, Bing; Kuramitsu, Howard K.

    2003-01-01

    In Streptococcus mutans, enzyme IIscr and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose. The scr regulon of S. mutans is composed of three genes, scrA and scrB, which code for enzyme IIscr and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator. It was previously shown that expression of both scrA and scrB is similarly induced by sucrose. Mutation in the scrR gene resulted ...

  9. The Transcriptional Repressor ID2 Can Interact with the Canonical Clock Components CLOCK and BMAL1 and Mediate Inhibitory Effects on mPer1 Expression*

    Ward, Sarah M.; Fernando, Shanik J.; Hou, Tim Y.; Duffield, Giles E.

    2010-01-01

    ID2 is a rhythmically expressed HLH transcriptional repressor. Deletion of Id2 in mice results in circadian phenotypes, highlighted by disrupted locomotor activity rhythms and an enhanced photoentrainment response. ID2 can suppress the transactivation potential of the positive elements of the clock, CLOCK-BMAL1, on mPer1 and clock-controlled gene (CCG) activity. Misregulation of CCGs is observed in Id2−/− liver, and mutant mice exhibit associated alterations in lipid homeostasis. These data s...

  10. Deubiquitylating Enzyme UBP64 Controls Cell Fate through Stabilization of the Transcriptional Repressor Tramtrack▿

    Bajpe, Prashanth Kumar; van der Knaap, Jan A.; Demmers, Jeroen A. A.; Bezstarosti, Karel; Bassett, Andrew; van Beusekom, Heleen M. M.; Travers, Andrew A.; Verrijzer, C. Peter

    2008-01-01

    Protein ubiquitylation plays a central role in multiple signal transduction pathways. However, the substrate specificity and potential developmental roles of deubiquitylating enzymes remain poorly understood. Here, we show that the Drosophila ubiquitin protease UBP64 controls cell fate in the developing eye. UBP64 represses neuronal cell fate but promotes the formation of nonneuronal cone cells. Using a proteomics approach, we identified the transcriptional repressor Tramtrack (TTK) as a primary UBP64 substrate. In common with TTK, reduced UBP64 levels lead to a loss of cone cells, supernumerary photoreceptors, and mechanosensory bristle cells. Previously, it was demonstrated that the blockade of neuronal cell fate was relieved by SINA-dependent ubiquitylation and degradation of TTK. We found that UBP64 counteracts SINA function by deubiquitylating TTK, leading to its stabilization and thereby promoting a nonneuronal cell fate. Mass spectrometric mapping revealed that SINA ubiquitylates multiple sites dispersed throughout TTK, which are duly deubiquitylated by UBP64. This observation suggests that both E3 SINA and UBP64 use a scanning mechanism to (de)ubiquitylate TTK. We conclude that the balance of TTK ubiquitylation by SINA and deubiquitylation by UBP64 constitutes a specific posttranslational switch controlling cell fate. PMID:18160715

  11. BigR, a Transcriptional Repressor from Plant-Associated Bacteria, Regulates an Operon Implicated in Biofilm Growth▿

    Barbosa, Rosicler L.; Benedetti, Celso E.

    2007-01-01

    Xylella fastidiosa is a plant pathogen that colonizes the xylem vessels, causing vascular occlusion due to bacterial biofilm growth. However, little is known about the molecular mechanisms driving biofilm formation in Xylella-plant interactions. Here we show that BigR (for “biofilm growth-associated repressor”) is a novel helix-turn-helix repressor that controls the transcription of an operon implicated in biofilm growth. This operon, which encodes BigR, membrane proteins, and an unusual beta...

  12. Transcriptional repressor Tbx3 is required for the hormone-sensing cell lineage in mammary epithelium.

    Kamini Kunasegaran

    Full Text Available The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production. Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.

  13. Repressor element-1 silencing transcription factor/neuronal restrictive silencer factor (REST/NRSF can regulate HSV-1 immediate-early transcription via histone modification

    Hill James M

    2007-06-01

    Full Text Available Abstract Background During primary infection of its human host, Herpes Simplex Virus Type-1 (HSV-1 establishes latency in neurons where the viral genome is maintained in a circular form associated with nucleosomes in a chromatin configration. During latency, most viral genes are silenced, although the molecular mechanisms responsible for this are unclear. We hypothesized that neuronal factors repress HSV-1 gene expression during latency. A search of the HSV-1 DNA sequence for potential regulatory elements identified a Repressor Element-1/Neuronal Restrictive Silencer Element (RE-1/NRSE located between HSV-1 genes ICP22 and ICP4. We predicted that the Repressor Element Silencing Transcription Factor/Neuronal Restrictive Silencer Factor (REST/NRSF regulates expression of ICP22 and ICP4. Results Transient cotransfection indicated that REST/NRSF inhibited the activity of both promoters. In contrast, cotransfection of a mutant form of REST/NRSF encoding only the DNA-binding domain of the protein resulted in less inhibition. Stably transformed cell lines containing episomal reporter plasmids with a chromatin structure showed that REST/NRSF specifically inhibited the ICP4 promoter, but not the ICP22 promoter. REST/NRSF inhibition of the ICP4 promoter was reversed by histone deacetylase (HDAC inhibitor Trichostatin A (TSA. Additionally, chromatin immuno-precipitation (ChIP assays indicated that the corepressor CoREST was recruited to the proximity of ICP4 promoter and that acetylation of histone H4 was reduced in the presence of REST/NRSF. Conclusion Since the ICP4 protein is a key transactivator of HSV-1 lytic cycle genes, these results suggest that REST/NRSF may have an important role in the establishment and/or maintenance of HSV-1 gene silencing during latency by targeting ICP4 expression.

  14. Identification of a Portable Repression Domain and an E1A-Responsive Activation Domain in Pax4: a Possible Role of Pax4 as a Transcriptional Repressor in the Pancreas

    Fujitani, Yoshio; Kajimoto, Yoshitaka; Yasuda, Tetsuyuki; Matsuoka, Taka-aki; Kaneto, Hideaki; Umayahara, Yutaka; Fujita, Noriko; Watada, Hirotaka; Miyazaki, Jun-ichi; Yamasaki, Yoshimitsu; Hori, Masatsugu

    1999-01-01

    Pax4 is a paired-domain (PD)-containing transcription factor which plays a crucial role in pancreatic β/δ-cell development. In this study, we characterized the DNA-binding and transactivation properties of mouse Pax4. Repetitive rounds of PCR-based selection led to identification of the optimal DNA-binding sequences for the PD of Pax4. In agreement with the conservation of the optimal binding sequences among the Pax family transcription factors, Pax4 could bind to the potential binding sites ...

  15. ANKRD1 acts as a transcriptional repressor of MMP13 via the AP-1 site.

    Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E; Davidson, Jeffrey M

    2014-04-01

    The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1(-/-) (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1(fl/fl) (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

  16. Negative regulation of the Wnt–β-catenin pathway by the transcriptional repressor HBP1

    Sampson, Ellen M.; Haque, Zaffar K.; Ku, Man-Ching; Tevosian, Sergei G; Albanese, Chris; Pestell, Richard G.; Paulson, K. Eric; Yee, Amy S.

    2001-01-01

    In certain cancers, constitutive Wnt signaling results from mutation in one or more pathway components. The result is the accumulation and nuclear localization of β-catenin, which interacts with the lymphoid enhancer factor-1 (LEF)/T-cell factor (TCF) family of HMG-box transcription factors, which activate important growth regulatory genes, including cyclin D1 and c-myc. As exemplified by APC and axin, the negative regulation of β-catenin is important for tumor suppression. Another potential ...

  17. Diploidy of Drosophila imaginal cells is maintained by a transcriptional repressor encoded by escargot.

    Fuse, N; Hirose, S; Hayashi, S

    1994-10-01

    The Drosophila escargot (esg) gene encodes a C2-H2-type zinc finger protein that is expressed in the imaginal discs and histoblasts. In some esg mutants, the abdominal histoblasts become polyploid. It has therefore been suggested that the role of esg is to maintain diploidy of the imaginal cells. We show that esg encodes a DNA-binding protein with high affinity for G/ACAGGTG, the consensus-binding sequence for the basic helix-loop-helix (bHLH) family of transcription factors (E2 box). This DNA-binding activity is essential for esg function in vivo as the strong embryonic lethal allele esgVS8 is caused by an amino acid change within the zinc finger region, leading to reduced affinity for DNA. In cultured cells, a heterodimer of the bHLH proteins Scute and Daughterless activates transcription from promoters containing E2 boxes. The esg protein strongly inhibits this activation, suggesting that esg may regulate developmental processes dependent on bHLH proteins. In larvae, esg protein expressed by the heat shock promoter can rescue the polyploid phenotype of abdominal histoblasts, demonstrating that the phenotype is attributable to a loss of esg function. esg must be expressed continuously during the larval period for efficient rescue. Ectopic expression of esg in the salivary glands inhibits endoreplication of DNA. These results suggest that esg is involved in transcriptional inhibition of genes required for endoreplication. PMID:7958894

  18. Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties

    Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties

  19. The src Homology 3-Like Domain of the Diphtheria Toxin Repressor (DtxR) Modulates Repressor Activation through Interaction with the Ancillary Metal Ion-Binding Site

    Love, John F.; vanderSpek, Johanna C.; Murphy, John R.

    2003-01-01

    The diphtheria toxin repressor (DtxR) is a transition metal ion-activated repressor that acts as a global regulatory element in the control of iron-sensitive genes in Corynebacterium diphtheriae. We recently described (L. Sun, J. C. vanderSpek, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 95:14985-14990, 1998) the isolation and in vivo characterization of a hyperactive mutant of DtxR, DtxR(E175K), that appeared to be constitutively active. We demonstrate here that while DtxR(E175K) remains ac...

  20. Transcriptional repressor domain of MBD1 is intrinsically disordered and interacts with its binding partners in a selective manner.

    Hameed, Umar Farook Shahul

    2014-05-09

    Methylation of DNA CpG sites is a major mechanism of epigenetic gene silencing and plays important roles in cell division, development and carcinogenesis. One of its regulators is the 64-residue C-terminal Transcriptional Repressor Domain (the TRD) of MBD1, which recruits several repressor proteins such as MCAF1, HDAC3 and MPG that are essential for the gene silencing. Using NMR spectroscopy, we have characterized the solution structure of the C-terminus of MBD1 (MBD1-c, residues D507 to Q605), which included the TRD (A529 to P592). Surprisingly, the MBD1-c is intrinsically disordered. Despite its lack of a tertiary folding, MBD1-c could still bind to different partner proteins in a selective manner. MPG and MCAF1Δ8 showed binding to both the N-terminal and C-terminal residues of MBD1-c but HDAC3 preferably bound to the C-terminal region. This study reveals how MBD1-c discriminates different binding partners, and thus, expands our understanding of the mechanisms of gene regulation by MBD1.

  1. The ZEB1 transcription factor is a novel repressor of adiposity in female mice.

    Jessica N Saykally

    Full Text Available BACKGROUND: Four genome-wide association studies mapped an "obesity" gene to human chromosome 10p11-12. As the zinc finger E-box binding homeobox 1 (ZEB1 transcription factor is encoded by the TCF8 gene located in that region, and as it influences the differentiation of various mesodermal lineages, we hypothesized that ZEB1 might also modulate adiposity. The goal of these studies was to test that hypothesis in mice. METHODOLOGY/PRINCIPAL FINDINGS: To ascertain whether fat accumulation affects ZEB1 expression, female C57BL/6 mice were fed a regular chow diet (RCD ad libitum or a 25% calorie-restricted diet from 2.5 to 18.3 months of age. ZEB1 mRNA levels in parametrial fat were six to ten times higher in the obese mice. To determine directly whether ZEB1 affects adiposity, wild type (WT mice and mice heterozygous for TCF8 (TCF8+/- were fed an RCD or a high-fat diet (HFD (60% calories from fat. By two months of age on an HFD and three months on an RCD, TCF8+/- mice were heavier than WT controls, which was attributed by Echo MRI to increased fat mass (at three months on an HFD: 0.517+/-0.081 total fat/lean mass versus 0.313+/-0.036; at three months on an RCD: 0.175+/-0.013 versus 0.124+/-0.012. No differences were observed in food uptake or physical activity, suggesting that the genotypes differ in some aspect of their metabolic activity. ZEB1 expression also increases during adipogenesis in cell culture. CONCLUSION/SIGNIFICANCE: These results show for the first time that the ZEB1 transcription factor regulates the accumulation of adipose tissue. Furthermore, they corroborate the genome-wide association studies that mapped an "obesity" gene at chromosome 10p11-12.

  2. Haploinsufficiency of MeCP2-interacting transcriptional co-repressor SIN3A causes mild intellectual disability by affecting the development of cortical integrity

    Witteveen, Josefine S; Willemsen, Marjolein H; Dombroski, Thaís C D; van Bakel, Nick H M; Nillesen, Willy M; van Hulten, Josephus A; Jansen, Eric J R; Verkaik, Dave; Veenstra-Knol, Hermine E.; Ravenswaaij-Arts, van Conny; Klein Wassink-Ruiter, Jolien S.; Vincent, Marie; David, Albert; Le Caignec, Cedric; Schieving, Jolanda; Gilissen, Christian; Foulds, Nicola; Rump, Patrick; Strom, Tim; Cremer, Kirsten; Zink, Alexander M; Engels, Hartmut; de Munnik, Sonja A; Visser, Jasper E; Brunner, Han G; Martens, Gerard J M; Pfundt, Rolph; Kleefstra, Tjitske; Kolk, Sharon M

    2016-01-01

    Numerous genes are associated with neurodevelopmental disorders such as intellectual disability and autism spectrum disorder (ASD), but their dysfunction is often poorly characterized. Here we identified dominant mutations in the gene encoding the transcriptional repressor and MeCP2 interactor switc

  3. Differential connectivity of splicing activators and repressors to the human spliceosome

    Akerman, Martin; Fregoso, Oliver I.; Das, Shipra; Ruse, Cristian; Jensen, Mads A.; Pappin, Darryl J.; Zhang, Michael Q.; Krainer, Adrian R.

    2015-01-01

    Background During spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions. Results Here, we investigate the protein connec...

  4. Loss of the transcriptional repressor PAG-3/Gfi-1 results in enhanced neurosecretion that is dependent on the dense-core vesicle membrane protein IDA-1/IA-2.

    Tao Cai

    2009-04-01

    Full Text Available It is generally accepted that neuroendocrine cells regulate dense core vesicle (DCV biogenesis and cargo packaging in response to secretory demands, although the molecular mechanisms of this process are poorly understood. One factor that has previously been implicated in DCV regulation is IA-2, a catalytically inactive protein phosphatase present in DCV membranes. Our ability to directly visualize a functional, GFP-tagged version of an IA-2 homolog in live Caenorhabditis elegans animals has allowed us to capitalize on the genetics of the system to screen for mutations that disrupt DCV regulation. We found that loss of activity in the transcription factor PAG-3/Gfi-1, which functions as a repressor in many systems, results in a dramatic up-regulation of IDA-1/IA-2 and other DCV proteins. The up-regulation of DCV components was accompanied by an increase in presynaptic DCV numbers and resulted in phenotypes consistent with increased neuroendocrine secretion. Double mutant combinations revealed that these PAG-3 mutant phenotypes were dependent on wild type IDA-1 function. Our results support a model in which IDA-1/IA-2 is a critical element in DCV regulation and reveal a novel genetic link to PAG-3-mediated transcriptional regulation. To our knowledge, this is the first mutation identified that results in increased neurosecretion, a phenotype that has clinical implications for DCV-mediated secretory disorders.

  5. A repressor activator protein1 homologue from an oleaginous strain of Candida tropicalis increases storage lipid production in Saccharomyces cerevisiae.

    Chattopadhyay, Atrayee; Dey, Prabuddha; Barik, Amita; Bahadur, Ranjit P; Maiti, Mrinal K

    2015-06-01

    The repressor activator protein1 (Rap1) has been studied over the years as a multifunctional regulator in Saccharomyces cerevisiae. However, its role in storage lipid accumulation has not been investigated. This report documents the identification and isolation of a putative transcription factor CtRap1 gene from an oleaginous strain of Candida tropicalis, and establishes the direct effect of its expression on the storage lipid accumulation in S. cerevisiae, usually a non-oleaginous yeast. In silico analysis revealed that the CtRap1 polypeptide binds relatively more strongly to the promoter of fatty acid synthase1 (FAS1) gene of S. cerevisiae than ScRap1. The expression level of CtRap1 transcript in vivo was found to correlate directly with the amount of lipid produced in oleaginous native host C. tropicalis. Heterologous expression of the CtRap1 gene resulted in ∼ 4-fold enhancement of storage lipid content (57.3%) in S. cerevisiae. We also showed that the functionally active CtRap1 upregulates the endogenous ScFAS1 and ScDGAT genes of S. cerevisiae, and this, in turn, might be responsible for the increased lipid production in the transformed yeast. Our findings pave the way for the possible utility of the CtRap1 gene in suitable microorganisms to increase their storage lipid content through transcription factor engineering. PMID:25805842

  6. A Comprehensive Catalog of Human KRAB-associated Zinc Finger Genes: Insights into the Evolutionary History of a Large Family of Transcriptional Repressors

    Huntley, S; Baggott, D M; Hamilton, A T; Tran-Gyamfi, M; Yang, S; Kim, J; Gordon, L; Branscomb, E; Stubbs, L

    2005-09-30

    Krueppel-type zinc finger (ZNF) motifs are prevalent components of transcription factor proteins in all eukaryotic species. In mammals, most ZNF proteins comprise a single class of transcriptional repressors in which a chromatin interaction domain, called the Krueppel-associated box (KRAB) is attached to a tandem array of DNA-binding zinc-finger motifs. KRAB-ZNF loci are specific to tetrapod vertebrates, but have expanded dramatically in numbers through repeated rounds of segmental duplication to create a gene family with hundreds of members in mammals. To define the full repertoire of human KRAB-ZNF proteins, we searched the human genome for key motifs and used them to construct and manually curate gene models. The resulting KRAB-ZNF gene catalog includes 326 known genes, 243 of which were structurally corrected by manual annotation, and 97 novel KRAB-ZNF genes; this single family therefore comprises 20% of all predicted human transcription factor genes. Many of the genes are alternatively spliced, yielding a total of 743 distinct predicted proteins. Although many human KRAB-ZNF genes are conserved in mammals, at least 136 and potentially more than 200 genes of this type are primate-specific including many recent segmental duplicates. KRAB-ZNF genes are active in a wide variety of human tissues suggesting roles in many key biological processes, but most member genes remain completely uncharacterized. Because of their sheer numbers, wide-ranging tissue-specific expression patterns, and remarkable evolutionary divergence we predict that KRAB-ZNF transcription factors have played critical roles in crafting many aspects of human biology, including both deeply conserved and primate-specific traits.

  7. Repressors report fewer intrusions following a laboratory stressor: the role of reduced stressor-relevant concept activation and inhibitory functioning.

    Overwijk, Sippie; Wessel, Ineke; de Jong, Peter J

    2009-03-01

    This study investigated whether a repressive coping style is associated with fewer intrusions following an experimentally controlled stressor. Furthermore, we examined whether lower activation of stressor-relevant concepts in long-term memory and better inhibitory functioning may contribute to this association. Extreme-scoring participants on a trait anxiety and a social desirability scale were selected to form repressor (n=35), low anxious (n=15), high anxious (n=30), and defensive (n=21) groups. In line with predictions, repressors reported fewer intrusions following a failure manipulation compared to non-repressors. Furthermore, pre-stressor inhibitory functioning was negatively associated with color-naming interference of stressor-related words. This suggests that overall, higher inhibitory control is related to lower activation of failure-related concepts. However, there was no evidence that concept activation and inhibitory control were responsible for repressors' lower number of self-reported intrusions. PMID:18937086

  8. The Transcription Repressor REST in Adult Neurons: Physiology, Pathology, and Diseases 1,2,3

    Baldelli, Pietro; Meldolesi, Jacopo

    2015-01-01

    Abstract REST [RE1-silencing transcription factor (also called neuron-restrictive silencer factor)] is known to repress thousands of possible target genes, many of which are neuron specific. To date, REST repression has been investigated mostly in stem cells and differentiating neurons. Current evidence demonstrates its importance in adult neurons as well. Low levels of REST, which are acquired during differentiation, govern the expression of specific neuronal phenotypes. REST-dependent genes...

  9. Regulation of MntH by a dual Mn(II- and Fe(II-dependent transcriptional repressor (DR2539 in Deinococcus radiodurans.

    Hongxing Sun

    Full Text Available The high intracellular Mn/Fe ratio observed within the bacteria Deinococcus radiodurans may contribute to its remarkable resistance to environmental stresses. We isolated DR2539, a novel regulator of intracellular Mn/Fe homeostasis in D. radiodurans. Electrophoretic gel mobility shift assays (EMSAs revealed that DR2539 binds specifically to the promoter of the manganese acquisition transporter (MntH gene, and that DR0865, the only Fur homologue in D. radiodurans, cannot bind to the promoter of mntH, but it can bind to the promoter of another manganese acquisition transporter, MntABC. β-galactosidase expression analysis indicated that DR2539 acts as a manganese- and iron-dependent transcriptional repressor. Further sequence alignment analysis revealed that DR2539 has evolved some special characteristics. Site-directed mutagenesis suggested that His98 plays an important role in the activities of DR2539, and further protein-DNA binding activity assays showed that the activity of H98Y mutants decreased dramatically relative to wild type DR2539. Our study suggests that D. radiodurans has evolved a very efficient manganese regulation mechanism that involves its high intracellular Mn/Fe ratio and permits resistance to extreme conditions.

  10. TGF-{beta} signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts

    Luciakova, Katarina, E-mail: katarina.luciakova@savba.sk [Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Vlarska 7, 833 91 Bratislava (Slovakia); Kollarovic, Gabriel; Kretova, Miroslava; Sabova, Ludmila [Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Vlarska 7, 833 91 Bratislava (Slovakia); Nelson, B. Dean [Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm (Sweden)

    2011-08-05

    Highlights: {yields} TGF-{beta} induces the formation of unique nuclear NF1/Smad4 complexes that repress expression of the ANT-2 gene. {yields} Repression is mediated through an NF1-dependent repressor element in the promoter. {yields} The formation of NF1/Smad4 complexes and the repression of ANT2 are prevented by inhibitors of p38 kinase and TGF-{beta} RI. {yields} NF1/Smad complexes implicate novel role for NF1 and Smad proteins in the regulation of growth. -- Abstract: We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G. Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123-130]. In the present study, we show that TGF-{beta}, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-{beta} is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-{beta} are prevented by inhibitors of TGF-{beta} RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-{beta} and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-{beta} signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.

  11. Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover.

    Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J; Shi, Yang; Harper, J Wade

    2013-10-10

    Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of parallel adaptor capture (PAC) proteomics and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

  12. ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes

    Peng, Haibing; TAN, LUJIAN; Osaki, Makoto; Zhan, Yumei; Ijiri, Kosei; Tsuchimochi, Kaneyuki; Otero, Miguel; Wang, Hong; CHOY, BOB K.; GRALL, FRANCK T.; Gu, Xuesong; Libermann, Towia A; Oettgen, Peter; Goldring, Mary B.

    2008-01-01

    The epithelium-specific ETS (ESE)-1 transcription factor is induced in chondrocytes by interleukin-1β (IL-1β). We reported previously that early activation of EGR-1 by IL-1β results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE-1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF-κB-dependent inhibition by IL-1β. Of the ETS factors tested, this response wa...

  13. The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS in Corynebacterium glutamicum

    Hartmann Michelle

    2007-11-01

    Full Text Available Abstract Background The major uptake system responsible for the transport of fructose, glucose, and sucrose in Corynebacterium glutamicum ATCC 13032 is the phosphoenolpyruvate:sugar phosphotransferase system (PTS. The genes encoding PTS components, namely ptsI, ptsH, and ptsF belong to the fructose-PTS gene cluster, whereas ptsG and ptsS are located in two separate regions of the C. glutamicum genome. Due to the localization within and adjacent to the fructose-PTS gene cluster, two genes coding for DeoR-type transcriptional regulators, cg2118 and sugR, are putative candidates involved in the transcriptional regulation of the fructose-PTS cluster genes. Results Four transcripts of the extended fructose-PTS gene cluster that comprise the genes sugR-cg2116, ptsI, cg2118-fruK-ptsF, and ptsH, respectively, were characterized. In addition, it was shown that transcription of the fructose-PTS gene cluster is enhanced during growth on glucose or fructose when compared to acetate. Subsequently, the two genes sugR and cg2118 encoding for DeoR-type regulators were mutated and PTS gene transcription was found to be strongly enhanced in the presence of acetate only in the sugR deletion mutant. The SugR regulon was further characterized by microarray hybridizations using the sugR mutant and its parental strain, revealing that also the PTS genes ptsG and ptsS belong to this regulon. Binding of purified SugR repressor protein to a 21 bp sequence identified the SugR binding site as an AC-rich motif. The two experimentally identified SugR binding sites in the fructose-PTS gene cluster are located within or downstream of the mapped promoters, typical for transcriptional repressors. Effector studies using electrophoretic mobility shift assays (EMSA revealed the fructose PTS-specific metabolite fructose-1-phosphate (F-1-P as a highly efficient, negative effector of the SugR repressor, acting in the micromolar range. Beside F-1-P, other sugar-phosphates like fructose

  14. Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113

    Abbas, Abdelhamid; John P. Morrissey; Marquez, Pilar Carnicero; Sheehan, Michelle M.; Delany, Isabel R.; O'Gara, Fergal

    2002-01-01

    The phlACBD genes responsible for the biosynthesis of the antifungal metabolite 2,4-diacetylphloroglucinol (PHL) by the biocontrol strain Pseudomonas fluorescens F113 are regulated at the transcriptional level by the pathway-specific repressor PhlF. Strong evidence suggests that this regulation occurs mainly in the early logarithmic phase of growth. First, the expression of the phlF gene is relatively high between 3 and 13 h of growth and relatively low thereafter, with the phlACBD operon fol...

  15. Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence

    Breslin, Mary B; Zhu, Min; Notkins, Abner L.; Lan, Michael S.

    2002-01-01

    A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/IA-1 fusion protein constructs, we demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the maj...

  16. The Developmental Regulator Protein Gon4l Associates with Protein YY1, Co-repressor Sin3a, and Histone Deacetylase 1 and Mediates Transcriptional Repression*

    Lu, Ping; Hankel, Isaiah L.; Hostager, Bruce S.; Swartzendruber, Julie A.; Friedman, Ann D.; Brenton, Janet L.; Rothman, Paul B.; Colgan, John D.

    2011-01-01

    Genetic studies involving zebrafish and mice have demonstrated that the protein Gon4l (Gon4-like) is essential for hematopoiesis. These studies also suggested that Gon4l regulates gene expression during hematopoietic development, yet the biochemical function of Gon4l has not been defined. Here, we describe the identification of factors that interact with Gon4l and may cooperate with this protein to regulate gene expression. As predicted by polypeptide sequence conservation, Gon4l interacted and co-localized with the DNA-binding protein YY1 (Yin Yang 1). Density gradient sedimentation analysis of protein lysates from mouse M12 B cells showed that Gon4l and YY1 co-sediment with the transcriptional co-repressor Sin3a and its functional partner histone deacetylase (HDAC) 1. Consistent with these results, immunoprecipitation studies showed that Gon4l associates with Sin3a, HDAC1, and YY1 as a part of complexes that form in M12 cells. Sequential immunoprecipitation studies demonstrated that Gon4l, YY1, Sin3a, and HDAC1 could all associate as components of a single complex and that a conserved domain spanning the central portion of Gon4l was required for formation of this complex. When targeted to DNA, Gon4l repressed the activity of a nearby promoter, which correlated with the ability to interact with Sin3a and HDAC1. Our data suggest that Sin3a, HDAC1, and YY1 are co-factors for Gon4l and that Gon4l may function as a platform for the assembly of complexes that regulate gene expression. PMID:21454521

  17. The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression.

    Lu, Ping; Hankel, Isaiah L; Hostager, Bruce S; Swartzendruber, Julie A; Friedman, Ann D; Brenton, Janet L; Rothman, Paul B; Colgan, John D

    2011-05-20

    Genetic studies involving zebrafish and mice have demonstrated that the protein Gon4l (Gon4-like) is essential for hematopoiesis. These studies also suggested that Gon4l regulates gene expression during hematopoietic development, yet the biochemical function of Gon4l has not been defined. Here, we describe the identification of factors that interact with Gon4l and may cooperate with this protein to regulate gene expression. As predicted by polypeptide sequence conservation, Gon4l interacted and co-localized with the DNA-binding protein YY1 (Yin Yang 1). Density gradient sedimentation analysis of protein lysates from mouse M12 B cells showed that Gon4l and YY1 co-sediment with the transcriptional co-repressor Sin3a and its functional partner histone deacetylase (HDAC) 1. Consistent with these results, immunoprecipitation studies showed that Gon4l associates with Sin3a, HDAC1, and YY1 as a part of complexes that form in M12 cells. Sequential immunoprecipitation studies demonstrated that Gon4l, YY1, Sin3a, and HDAC1 could all associate as components of a single complex and that a conserved domain spanning the central portion of Gon4l was required for formation of this complex. When targeted to DNA, Gon4l repressed the activity of a nearby promoter, which correlated with the ability to interact with Sin3a and HDAC1. Our data suggest that Sin3a, HDAC1, and YY1 are co-factors for Gon4l and that Gon4l may function as a platform for the assembly of complexes that regulate gene expression. PMID:21454521

  18. Activation of pur Gene Expression by a Homologue of the Bacillus subtilis PurR repressor:

    Kilstrup, Mogens; Martinussen, Jan

    1998-01-01

    R encoded repressor from Bacillus subtilis. The wildtype purR gene complements the purine auxotrophy of a purR::Iss1mutant, and it was shown that the purR::Iss1 mutation lowers transcription from the purine regulated L. lactis purD promoter. In a parallel study on the regulation of purC and purD expression....... We have identified a PurBox sequence overlapping the -35 region of the L. lactis purR promoter and found, by studies of a purR-lacLM fusion plasmid, that purR is autoregulated. Because of the high similarity of the PurR proteins from B. subtilis and L. lactis, we looked for PurBox sequences in the...... promoter regions of the PurR regulated genes in B. subtilis, and identified a perfectly matching PurBox in the purA promoter region, and slightly degenerate PurBox like sequences in the promoter regions for the pur operon and the purR gene....

  19. Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD+-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.

  20. Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    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.

  1. The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells

    Gullberg Urban; Dhanda Rakesh; Ajore Ram; Olsson Inge

    2010-01-01

    Abstract Background The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and...

  2. Mutant lambda phage repressor with a specific defect in its positive control function.

    Guarente, L; Nye, J. S.; Hochschild, A; Ptashne, M

    1982-01-01

    The lambda phage repressor is both a positive and a negative regulator of gene transcription. We describe a mutant lambda phage repressor that has specifically lost its activator function. The mutant binds to the lambda phage operator sites and represses the lambda phage promoters PR and PL. However, it fails to stimulate transcription from the promoter PRM. The mutation lies in that portion of repressor--namely, the amino-terminal domain--that has been shown [Sauer, R. T., Pabo, C. O., Meyer...

  3. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Goto, Yamafumi [Department of Dermatology, Shinshu University School of Medicine, Matsumoto (Japan); Takata, Minoru [Department of Dermatology, Okayama University Graduate School of Medical Dentistry and Pharmaceutical Sciences, Okayama (Japan); Turkson, James; Li, Xiaoman Shawn [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States); Zervos, Antonis S., E-mail: azervos@mail.ucf.edu [Biomolecular Science Center, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL (United States)

    2011-01-07

    Research highlights: {yields} THAP5 is a DNA-binding protein and a transcriptional repressor. {yields} THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. {yields} THAP5 induction correlates with the degree of apoptosis in melanoma cell population. {yields} THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

  4. THAP5 is a DNA-binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Research highlights: → THAP5 is a DNA-binding protein and a transcriptional repressor. → THAP5 is induced in melanoma cells upon exposure to UV or treatment with cisplatin. → THAP5 induction correlates with the degree of apoptosis in melanoma cell population. → THAP5 is a pro-apoptotic protein involved in melanoma cell death. -- Abstract: THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA-binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death.

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

    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

  6. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation

    Barbosa, Rosicler Lázaro; Rinaldi, Fábio Cupri; Guimarães, Beatriz Gomes, E-mail: beatriz@lnls.br; Benedetti, Celso Eduardo, E-mail: beatriz@lnls.br [Center for Molecular and Structural Biology, Brazilian Synchrotron Light Laboratory, Campinas, SP, CP 6192, CEP 13083-970 (Brazil)

    2007-07-01

    In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix–turn–helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 Å, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit.

  7. Crystallization and preliminary X-ray analysis of BigR, a transcription repressor from Xylella fastidiosa involved in biofilm formation

    In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. BigR (biofilm growth-associated repressor) is a novel repressor protein that regulates the transcription of an operon implicated in biofilm growth in both Xylella fastidiosa and Agrobacterium tumefaciens. This protein binds to a palindromic TA-rich element located in the promoter of the BigR operon and strongly represses transcription of the operon. BigR contains a helix–turn–helix (HTH) domain that is found in some members of the ArsR/SmtB family of metal sensors, which control metal resistance in bacteria. Although functional studies have suggested that BigR does not act as a metal sensor, the presence of two cysteines and a methionine in its primary structure raised the possibility of BigR being a metal-ligand protein. In order to gain new insights into the protein structure and its possible interaction with a metal ion or effector ligand, BigR from X. fastidiosa was crystallized in native and selenomethionine (SeMet) labelled forms using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from native and SeMet crystals to resolutions of 1.95 and 2.2 Å, respectively. Both crystals belong to space group P321 and contain one molecule per asymmetric unit

  8. Characterization of the Lactococcus lactis lactose operon promoter: contribution of flanking sequences and LacR repressor to promoter activity.

    van Rooijen, R J; Gasson, M. J.; de Vos, W M

    1992-01-01

    We determined the location, activity, and regulation of the promoter of the Lactococcus lactis 8-kb lactose operon (lacABCDFEGX), which encodes the enzymes of the lactose phosphotransferase system and the tagatose 6-phosphate pathway. The lac promoter sequence corresponds closely to the consensus promoter described for gram-positive bacteria and is located in a back-to-back configuration with the promoter of the divergently transcribed lacR gene, which encodes the LacR repressor. The transcri...

  9. An ABA down-regulated bHLH transcription repressor gene, bHLH129 regulates root elongation and ABA response when overexpressed in Arabidopsis

    Tian, Hainan; Guo, Hongyan; Dai, Xuemei; Cheng, Yuxin; Zheng, Kaijie; Wang, Xiaoping; Wang, Shucai

    2015-01-01

    Plant hormone abscisic acid (ABA) plays a crucial role in modulating plant responses to environmental stresses. Basic helix-loop-helix (bHLH) transcription factors are one of the largest transcription factor families that regulate multiple aspects of plant growth and development, as well as of plant metabolism in Arabidopsis. Several bHLH transcription factors have been shown to be involved in the regulation of ABA signaling. We report here the characterization of bHLH129, a bHLH transcription factor in Arabidopsis. We found that the expression level of bHLH129 was reduced in response to exogenously applied ABA, and elevated in the ABA biosynthesis mutant aba1-5. Florescence observation of transgenic plants expressing bHLH129-GFP showed that bHLH129 was localized in the nucleus, and transient expression of bHLH129 in protoplasts inhibited reporter gene expression. When expressed in Arabidopsis under the control of the 35S promoter, bHLH129 promoted root elongation, and the transgenic plants were less sensitivity to ABA in root elongation assays. Quantitative RT-PCR results showed that ABA response of several genes involved in ABA signaling, including ABI1, SnRK2.2, SnRK2.3 and SnRK2.6 were altered in the transgenic plants overexpressing bHLH129. Taken together, our study suggests that bHLH129 is a transcription repressor that negatively regulates ABA response in Arabidopsis. PMID:26625868

  10. The ETS domain transcriptional repressor Anterior open inhibits MAP kinase and Wingless signaling to couple tracheal cell fate with branch identity.

    Caviglia, Sara; Luschnig, Stefan

    2013-03-01

    Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. Here, we show that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. We demonstrate that Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network. PMID:23444354

  11. PRISM/PRDM6, a Transcriptional Repressor That Promotes the Proliferative Gene Program in Smooth Muscle Cells

    Davis, Christopher A.; Haberland, Michael; Arnold, Michael A.; Sutherland, Lillian B.; McDonald, Oliver G.; Richardson, James A.; Childs, Geoffrey; Harris, Stephen; Owens, Gary K.; Olson, Eric N.

    2006-01-01

    Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expr...

  12. THAP5 is a DNA binding transcriptional repressor that is regulated in melanoma cells during DNA damage-induced cell death

    Balakrishnan, Meenakshi P.; Cilenti, Lucia; Ambivero, Camilla; Goto, Yamafumi; Takata, Minoru; Turkson, James; Li, Xiaoman Shawn; Zervos, Antonis S.

    2011-01-01

    THAP5 was originally isolated as a specific interactor and substrate of the mitochondrial pro-apoptotic Omi/HtrA2 protease. It is a human zinc finger protein characterized by a restricted pattern of expression and the lack of orthologs in mouse and rat. The biological function of THAP5 is unknown but our previous studies suggest it could regulate G2/M transition in kidney cells and could be involved in human cardiomyocyte cell death associated with coronary artery disease (CAD). In this report, we expanded our studies on the properties and function of THAP5 in human melanoma cells. THAP5 was expressed in primary human melanocytes as well as in all melanoma cell lines that were tested. THAP5 protein level was significantly induced by UV irradiation or cisplatin treatment, conditions known to cause DNA damage. The induction of THAP5 correlated with a significant increase in apoptotic cell death. In addition, we show that THAP5 is a nuclear protein that could recognize and bind a specific DNA motif. THAP5 could also repress the transcription of a reporter gene in a heterologous system. Our work suggests that THAP5 is a DNA binding protein and a transcriptional repressor. Furthermore, THAP5 has a pro-apoptotic function and it was induced in melanoma cells under conditions that promoted cell death. PMID:21110952

  13. Lymphoid Progenitor Cells from Childhood Acute Lymphoblastic Leukemia Are Functionally Deficient and Express High Levels of the Transcriptional Repressor Gfi-1

    Jessica Purizaca

    2013-01-01

    Full Text Available Acute lymphoblastic leukemia (ALL is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34+ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

  14. Acyl-Acyl carrier protein regulates transcription of fatty acid biosynthetic genes via the FabT repressor in Streptococcus pneumoniae.

    Jerga, Agoston; Rock, Charles O

    2009-06-01

    Long-chain acyl-acyl carrier proteins (acyl-ACP) are established biochemical regulators of bacterial type II fatty acid synthases due to their ability to feedback-inhibit the early steps in the biosynthetic pathway. In Streptococcus pneumoniae, the expression of the fatty acid synthase (fab) genes is controlled by a helix-turn-helix transcriptional repressor called FabT. A screen of pathway intermediates identified acyl-ACP as a ligand that increased the affinity of FabT for DNA. FabT bound to a wide range of acyl-ACP chain lengths in the absence of DNA, but only the long-chain acyl-ACPs increase the affinity of FabT for DNA. FabT affinity for DNA increased with increasing acyl-ACP chain length with cis-vaccenoyl-ACP being the most effective ligand. Thus, FabT is a new ACP-interacting partner that acts as a transcriptional rheostat to fine tune the expression of the fab genes based on the demand for fatty acids. PMID:19376778

  15. Acyl-Acyl Carrier Protein Regulates Transcription of Fatty Acid Biosynthetic Genes via the FabT Repressor in Streptococcus pneumoniae*

    Jerga, Agoston; Rock, Charles O.

    2009-01-01

    Long-chain acyl-acyl carrier proteins (acyl-ACP) are established biochemical regulators of bacterial type II fatty acid synthases due to their ability to feedback-inhibit the early steps in the biosynthetic pathway. In Streptococcus pneumoniae, the expression of the fatty acid synthase (fab) genes is controlled by a helix-turn-helix transcriptional repressor called FabT. A screen of pathway intermediates identified acyl-ACP as a ligand that increased the affinity of FabT for DNA. FabT bound to a wide range of acyl-ACP chain lengths in the absence of DNA, but only the long-chain acyl-ACPs increase the affinity of FabT for DNA. FabT affinity for DNA increased with increasing acyl-ACP chain length with cis-vaccenoyl-ACP being the most effective ligand. Thus, FabT is a new ACP-interacting partner that acts as a transcriptional rheostat to fine tune the expression of the fab genes based on the demand for fatty acids. PMID:19376778

  16. Haploinsufficiency of MeCP2-interacting transcriptional co-repressor SIN3A causes mild intellectual disability by affecting the development of cortical integrity.

    Witteveen, Josefine S; Willemsen, Marjolein H; Dombroski, Thaís C D; van Bakel, Nick H M; Nillesen, Willy M; van Hulten, Josephus A; Jansen, Eric J R; Verkaik, Dave; Veenstra-Knol, Hermine E; van Ravenswaaij-Arts, Conny M A; Wassink-Ruiter, Jolien S Klein; Vincent, Marie; David, Albert; Le Caignec, Cedric; Schieving, Jolanda; Gilissen, Christian; Foulds, Nicola; Rump, Patrick; Strom, Tim; Cremer, Kirsten; Zink, Alexander M; Engels, Hartmut; de Munnik, Sonja A; Visser, Jasper E; Brunner, Han G; Martens, Gerard J M; Pfundt, Rolph; Kleefstra, Tjitske; Kolk, Sharon M

    2016-08-01

    Numerous genes are associated with neurodevelopmental disorders such as intellectual disability and autism spectrum disorder (ASD), but their dysfunction is often poorly characterized. Here we identified dominant mutations in the gene encoding the transcriptional repressor and MeCP2 interactor switch-insensitive 3 family member A (SIN3A; chromosome 15q24.2) in individuals who, in addition to mild intellectual disability and ASD, share striking features, including facial dysmorphisms, microcephaly and short stature. This phenotype is highly related to that of individuals with atypical 15q24 microdeletions, linking SIN3A to this microdeletion syndrome. Brain magnetic resonance imaging showed subtle abnormalities, including corpus callosum hypoplasia and ventriculomegaly. Intriguingly, in vivo functional knockdown of Sin3a led to reduced cortical neurogenesis, altered neuronal identity and aberrant corticocortical projections in the developing mouse brain. Together, our data establish that haploinsufficiency of SIN3A is associated with mild syndromic intellectual disability and that SIN3A can be considered to be a key transcriptional regulator of cortical brain development. PMID:27399968

  17. Transcriptional regulation of the presenilin-1 gene controls gamma-secretase activity.

    Lee, Sebum; Das, Hriday K

    2010-01-01

    Inhibition of basal JNK activity by JNK inhibitor SP600125 or JNK1siRNA repressed presenilin-1 (PS1) expression in SK-N-SH cells by augmenting the level of p53, a repressor of the PS1 gene (1). We now showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited gamma-secretase mediated processing of amyloid precursor protein (APP) resulting in the accumulation of C99 fragment and the reduction of secreted Abeta40 level without altering the expression of nicastrin (NCT). Co-treatment of cells with SP600125 and p53 inhibitor, pifithrin-alpha, partially nullified the suppressive effects of SP610025 on PS1 expression and secreted Abeta40 level. Suppression of JNK1 by JNK1siRNA also decreased Abeta40 level. Furthermore, overexpression of the repressors p53, ZNF237 and CHD3 of the PS1 gene also suppressed the processing of APP through repression of PS1 transcription by deacetylation of histone at the PS1 promoter. Transcriptional activator Ets2 increased PS1 protein and secreted Abeta40 levels without affecting the expression of NCT by activating PS1 transcription via hyper-acetylation of histone at the PS1 promoter. Therefore, regulation of PS1 transcription modulates gamma-secretase activity. PMID:20036849

  18. TBLR1 regulates the expression of nuclear hormone receptor co-repressors

    Brown Stuart

    2006-08-01

    Full Text Available Abstract Background Transcription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes. Results TBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies ~200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of

  19. Intrinsic transcript cleavage activity of RNA polymerase.

    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...

  20. The transcriptional repressor TupA in Aspergillus niger is involved in controlling gene expression related to cell wall biosynthesis, development, and nitrogen source availability.

    Doreen Schachtschabel

    Full Text Available The Tup1-Cyc8 (Ssn6 complex is a well characterized and conserved general transcriptional repressor complex in eukaryotic cells. Here, we report the identification of the Tup1 (TupA homolog in the filamentous fungus Aspergillus niger in a genetic screen for mutants with a constitutive expression of the agsA gene. The agsA gene encodes a putative alpha-glucan synthase, which is induced in response to cell wall stress in A. niger. Apart from the constitutive expression of agsA, the selected mutant was also found to produce an unknown pigment at high temperatures. Complementation analysis with a genomic library showed that the tupA gene could complement the phenotypes of the mutant. Screening of a collection of 240 mutants with constitutive expression of agsA identified sixteen additional pigment-secreting mutants, which were all mutated in the tupA gene. The phenotypes of the tupA mutants were very similar to the phenotypes of a tupA deletion strain. Further analysis of the tupA-17 mutant and the ΔtupA mutant revealed that TupA is also required for normal growth and morphogenesis. The production of the pigment at 37°C is nitrogen source-dependent and repressed by ammonium. Genome-wide expression analysis of the tupA mutant during exponential growth revealed derepression of a large group of diverse genes, including genes related to development and cell wall biosynthesis, and also protease-encoding genes that are normally repressed by ammonium. Comparison of the transcriptome of up-regulated genes in the tupA mutant showed limited overlap with the transcriptome of caspofungin-induced cell wall stress-related genes, suggesting that TupA is not a general suppressor of cell wall stress-induced genes. We propose that TupA is an important repressor of genes related to development and nitrogen metabolism.

  1. Mechanism of Iron-Dependent Repressor (IdeR) Activation and DNA Binding: A Molecular Dynamics and Protein Structure Network Study

    Ghosh, Soma; Chandra, Nagasuma; Vishveshwara, Saraswathi

    2015-01-01

    Metalloproteins form a major class of enzymes in the living system that are involved in crucial biological functions such as catalysis, redox reactions and as ‘switches’ in signal transductions. Iron dependent repressor (IdeR) is a metal-sensing transcription factor that regulates free iron concentration in Mycobacterium tuberculosis. IdeR is also known to promote bacterial virulence, making it an important target in the field of therapeutics. Mechanistic details of how iron ions modulate Ide...

  2. Mechanism of Iron-Dependent Repressor (IdeR) Activation and DNA Binding: A Molecular Dynamics and Protein Structure Network Study.

    Soma Ghosh; Nagasuma Chandra; Saraswathi Vishveshwara

    2015-01-01

    Metalloproteins form a major class of enzymes in the living system that are involved in crucial biological functions such as catalysis, redox reactions and as 'switches' in signal transductions. Iron dependent repressor (IdeR) is a metal-sensing transcription factor that regulates free iron concentration in Mycobacterium tuberculosis. IdeR is also known to promote bacterial virulence, making it an important target in the field of therapeutics. Mechanistic details of how iron ions modulate Ide...

  3. Initiation binding repressor, a factor that binds to the transcription initiation site of the histone h5 gene, is a glycosylated member of a family of cell growth regulators [corrected

    Gómez-Cuadrado, A; Martín, M; Noël, M; Ruiz-Carrillo, A

    1995-01-01

    Initiation binding repressor [corrected] (IBR) is a chicken erythrocyte factor (apparent molecular mass, 70 to 73 kDa) that binds to the sequences spanning the transcription initiation site of the histone h5 gene, repressing its transcription. A variety of other cells, including transformed erythroid precursors, do not have IBR but a factor referred to as IBF (68 to 70 kDa) that recognizes the same IBR sites. We have cloned the IBR cDNA and studied the relationship of IBR and IBF. IBR is a 50...

  4. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    Research highlights: → High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. → Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. → Rbpj upregulated promoter activities of Runx2 and Ose2. → Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

  5. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    Wang, Shengchao [Department of Preventive Dentistry, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, 710032 Xi' an (China); Kawashima, Nobuyuki, E-mail: kawashima.n.endo@tmd.ac.jp [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Sakamoto, Kei; Katsube, Ken-ichi [Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Umezawa, Akihiro [Department of Reproductive Biology and Pathology, National Institute for Child Health and Development, 2-10-4 Ohkura, Setagaya-ku, Tokyo 157-8535 (Japan); Suda, Hideaki [Department of Pulp Biology and Endodontics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); GCOE Program, International Research Center for Molecular Science in Tooth and Bone Diseases, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    2010-09-10

    Research highlights: {yields} High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. {yields} Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. {yields} Rbpj upregulated promoter activities of Runx2 and Ose2. {yields} Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.

  6. A POP-1 repressor complex restricts inappropriate cell type-specific gene transcription during Caenorhabditis elegans embryogenesis

    Calvo, Dominica; Victor, Martin; Gay, Frédérique; Sui, Guangchao; Luke, Margaret Po-Shan; Dufourcq, Pascale; Wen, Gengyun; Maduro, Morris; Rothman, Joel; Shi, Yang

    2001-01-01

    In Caenorhabditis elegans, histone acetyltransferase CBP-1 counteracts the repressive activity of the histone deacetylase HDA-1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box-containing protein, POP-1, through an unknown mechanism. In this study, we show that CBP-1, HDA-1 and POP-1 converge on end-1, an initial endoderm-determining gene. In the E lineage, an essential function of CBP-1 appe...

  7. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated IG{epsilon} gene transcription by recruitment of HDAC1

    Dong, Lijie; Zhang, Xinyu; Fu, Xiao;

    2010-01-01

    Regulation of transcription requires cooperation between sequence specific transcription factors and numerous coregulatory proteins. In IL-4/IL-13 signaling several coactivators for STAT6 have been identified, but the molecular mechanisms of STAT6-mediated gene transcription are still not fully...... understood. Here we identified by proteomic approach that PTB-associated splicing factor (PSF) interacts with STAT6. In cells the interaction required IL-4 stimulation and was observed both with endogenous and ectopically expressed proteins. The ligand dependency of the interaction suggested involvement of...... phosphorylation, and IL-4 stimulation increased tyrosine phosphorylation of PSF and STAT6. Functional analysis demonstrated that ectopic expression of PSF resulted in inhibition of STAT6-mediated gene transcriptional activation and mRNA expression of Ig heavy chain germline Ig ε, while knockdown of PSF increased...

  8. Mechanism of Iron-Dependent Repressor (IdeR) Activation and DNA Binding: A Molecular Dynamics and Protein Structure Network Study.

    Ghosh, Soma; Chandra, Nagasuma; Vishveshwara, Saraswathi

    2015-12-01

    Metalloproteins form a major class of enzymes in the living system that are involved in crucial biological functions such as catalysis, redox reactions and as 'switches' in signal transductions. Iron dependent repressor (IdeR) is a metal-sensing transcription factor that regulates free iron concentration in Mycobacterium tuberculosis. IdeR is also known to promote bacterial virulence, making it an important target in the field of therapeutics. Mechanistic details of how iron ions modulate IdeR such that it dimerizes and binds to DNA is not understood clearly. In this study, we have performed molecular dynamic simulations and integrated it with protein structure networks to study the influence of iron on IdeR structure and function. A significant structural variation between the metallated and the non-metallated system is observed. Our simulations clearly indicate the importance of iron in stabilizing its monomeric subunit, which in turn promotes dimerization. However, the most striking results are obtained from the simulations of IdeR-DNA complex in the absence of metals, where at the end of 100ns simulations, the protein subunits are seen to rapidly dissociate away from the DNA, thereby forming an excellent resource to investigate the mechanism of DNA binding. We have also investigated the role of iron as an allosteric regulator of IdeR that positively induces IdeR-DNA complex formation. Based on this study, a mechanistic model of IdeR activation and DNA binding has been proposed. PMID:26699663

  9. Mechanism of Iron-Dependent Repressor (IdeR Activation and DNA Binding: A Molecular Dynamics and Protein Structure Network Study.

    Soma Ghosh

    2015-12-01

    Full Text Available Metalloproteins form a major class of enzymes in the living system that are involved in crucial biological functions such as catalysis, redox reactions and as 'switches' in signal transductions. Iron dependent repressor (IdeR is a metal-sensing transcription factor that regulates free iron concentration in Mycobacterium tuberculosis. IdeR is also known to promote bacterial virulence, making it an important target in the field of therapeutics. Mechanistic details of how iron ions modulate IdeR such that it dimerizes and binds to DNA is not understood clearly. In this study, we have performed molecular dynamic simulations and integrated it with protein structure networks to study the influence of iron on IdeR structure and function. A significant structural variation between the metallated and the non-metallated system is observed. Our simulations clearly indicate the importance of iron in stabilizing its monomeric subunit, which in turn promotes dimerization. However, the most striking results are obtained from the simulations of IdeR-DNA complex in the absence of metals, where at the end of 100ns simulations, the protein subunits are seen to rapidly dissociate away from the DNA, thereby forming an excellent resource to investigate the mechanism of DNA binding. We have also investigated the role of iron as an allosteric regulator of IdeR that positively induces IdeR-DNA complex formation. Based on this study, a mechanistic model of IdeR activation and DNA binding has been proposed.

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

    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

  11. Surface expression of GABAA receptors is transcriptionally controlled by the interplay of cAMP-response element-binding protein and its binding partner inducible cAMP early repressor.

    Hu, Yinghui; Lund, Ingrid V; Gravielle, Maria C; Farb, David H; Brooks-Kayal, Amy R; Russek, Shelley J

    2008-04-01

    The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers. PMID:18180303

  12. Structural mechanism of transcription regulation of the Staphylococcus aureus multidrug efflux operon mepRA by the MarR family repressor MepR.

    Birukou, Ivan; Seo, Susan M; Schindler, Bryan D; Kaatz, Glenn W; Brennan, Richard G

    2014-02-01

    The multidrug efflux pump MepA is a major contributor to multidrug resistance in Staphylococcus aureus. MepR, a member of the multiple antibiotic resistance regulator (MarR) family, represses mepA and its own gene. Here, we report the structure of a MepR-mepR operator complex. Structural comparison of DNA-bound MepR with 'induced' apoMepR reveals the large conformational changes needed to allow the DNA-binding winged helix-turn-helix motifs to interact with the consecutive major and minor grooves of the GTTAG signature sequence. Intriguingly, MepR makes no hydrogen bonds to major groove nucleobases. Rather, recognition-helix residues Thr60, Gly61, Pro62 and Thr63 make sequence-specifying van der Waals contacts with the TTAG bases. Removing these contacts dramatically affects MepR-DNA binding activity. The wings insert into the flanking minor grooves, whereby residue Arg87, buttressed by Asp85, interacts with the O2 of T4 and O4' ribosyl oxygens of A23 and T4. Mutating Asp85 and Arg87, both conserved throughout the MarR family, markedly affects MepR repressor activity. The His14':Arg59 and Arg10':His35:Phe108 interaction networks stabilize the DNA-binding conformation of MepR thereby contributing significantly to its high affinity binding. A structure-guided model of the MepR-mepA operator complex suggests that MepR dimers do not interact directly and cooperative binding is likely achieved by DNA-mediated allosteric effects. PMID:24293644

  13. LacR is a repressor of lacABCD and LacT is an activator of lacTFEG, constituting the lac gene cluster in Streptococcus pneumoniae.

    Afzal, Muhammad; Shafeeq, Sulman; Kuipers, Oscar P

    2014-09-01

    Comparison of the transcriptome of Streptococcus pneumoniae strain D39 grown in the presence of either lactose or galactose with that of the strain grown in the presence of glucose revealed the elevated expression of various genes and operons, including the lac gene cluster, which is organized into two operons, i.e., lac operon I (lacABCD) and lac operon II (lacTFEG). Deletion of the DeoR family transcriptional regulator lacR that is present downstream of the lac gene cluster revealed elevated expression of lac operon I even in the absence of lactose. This suggests a function of LacR as a transcriptional repressor of lac operon I, which encodes enzymes involved in the phosphorylated tagatose pathway in the absence of lactose or galactose. Deletion of lacR did not affect the expression of lac operon II, which encodes a lactose-specific phosphotransferase. This finding was further confirmed by β-galactosidase assays with PlacA-lacZ and PlacT-lacZ in the presence of either lactose or glucose as the sole carbon source in the medium. This suggests the involvement of another transcriptional regulator in the regulation of lac operon II, which is the BglG-family transcriptional antiterminator LacT. We demonstrate the role of LacT as a transcriptional activator of lac operon II in the presence of lactose and CcpA-independent regulation of the lac gene cluster in S. pneumoniae. PMID:24951784

  14. Acidic phospholipid-independent interaction of Yas3p, an Opi1-family transcriptional repressor of Yarrowia lipolytica, with the endoplasmic reticulum.

    Kobayashi, Satoshi; Tezaki, Satoshi; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori

    2015-12-01

    In the n-alkane-assimilating yeast Yarrowia lipolytica, the transcription of ALK1, encoding cytochrome P450, that catalyses n-alkane hydroxylation is activated by a complex composed of Yas1p and Yas2p via a promoter element, ARE1, in response to n-alkanes. An Opi1-family transcription factor, Yas3p, represses the transcription by binding to Yas2p in the nucleus when cultured in glucose-containing medium, but it is localized to the ER, presumably through interaction with acidic phospholipids, phosphatidic acid and/or phospho inositides, when cultured in n-alkane-containing medium. Here, to elucidate the mechanisms regulating the localization of Yas3p, point and deletion mutants of Yas3p were constructed and analysed. The substitution of Trp(360) and Cys(361) by Arg abrogated the localization of Yas3p to the ER and decreased ARE1-mediated transcriptional activation by n-alkane. A Yas3p truncation mutant consisting of residues 259-422 did not bind to acidic phospholipids, but it was localized to the ER in the presence of n-alkane, implying the acidic-phospholipid-independent recruitment of this mutant to the ER in response to n-alkane. The W360R and C361R substitutions in this truncation mutant abolished its localization to the ER. The results suggest that these residues are implicated in the acidic phospholipid-independent interaction of Yas3p to the ER. PMID:26284565

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

    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.

  16. The activation function 2 domain of hepatic nuclear factor 4 is regulated by a short C-terminal proline-rich repressor domain.

    Iyemere, V P; Davies, N H; Brownlee, G G

    1998-01-01

    Hepatic nuclear factor 4 (HNF4) is a transcription factor whose expression is crucial for mouse embryonic development, for liver-specific gene expression and for the prevention of one form of maturity-onset diabetes of the young. Its domain structure has been defined previously and is similar to other members of the nuclear receptor superfamily. A repressor domain has now been localised to a region of 14 amino acids (residues 428-441) near the C-terminus of HNF4 and is sufficient by itself to...

  17. Poly(ADP-Ribose) Polymerase 1 and Ste20-Like Kinase hKFC Act as Transcriptional Repressors for Gamma-2 Herpesvirus Lytic Replication

    Gwack, Yousang; Nakamura, Hiroyuki; Lee, Sun Hwa; Souvlis, John; Yustein, Jason T.; Gygi, Steve; Kung, Hsing-Jien; Jung, Jae U.

    2003-01-01

    The replication and transcription activator (RTA) of gamma-2 herpesvirus is sufficient to drive the entire virus lytic cycle. Hence, the control of RTA activity should play an important role in the maintenance of viral latency. Here, we demonstrate that cellular poly(ADP-ribose) polymerase 1 (PARP-1) and Ste20-like kinase hKFC interact with the serine/threonine-rich region of gamma-2 herpesvirus RTA and that these interactions efficiently transfer poly(ADP-ribose) and phosphate units to RTA. ...

  18. Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1*S⃞

    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...

  19. STENOFOLIA acts as a repressor in regulating leaf blade outgrowth.

    Lin, Hao; Niu, Lifang; Tadege, Million

    2013-06-01

    We recently reported that the Medicago WOX gene, STENOFOLIA (STF), acts as a transcriptional repressor in regulating leaf blade outgrowth. By using the Nicotiana sylvestris bladeless lam1 mutant as a genetic tool, we showed that the WUS-box, which is conserved among WUS clade WOX genes, is partly responsible for the repressive activity of STF. All members of the modern/WUS clade genes (WUS, WOX1-WOX7) in Arabidopsis that contain intact WUS-box can substitute for STF/LAM1 function while the intermediate and ancient clade members including WOX9,WOX11 and WOX13 cannot, due to lack of the intact WUS-box. Taken together, our results reveal a conserved repression mechanism playing a central role in cell proliferation conferred to the evolutionarily dynamic WOX gene family with acquisition of a repressor domain. PMID:23603965

  20. Proto-oncogene FBI-1 (Pokemon/ZBTB7A) represses transcription of the tumor suppressor Rb gene via binding competition with Sp1 and recruitment of co-repressors.

    Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

    2008-11-28

    FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp -308 to -188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp -65 to -56) and GC-box 2 (bp -18 to -9), the latter of which is also bound by FBI-1. We found that FRE3 (bp -244 to -236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression. PMID:18801742

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

    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

  2. Bovine papillomavirus type 1 E2 transcriptional regulators directly bind two cellular transcription factors, TFIID and TFIIB.

    Rank, N M; Lambert, P F

    1995-01-01

    The bovine papillomavirus type 1 (BPV-1) E2 translational open reading frame encodes three proteins that regulate viral transcription and DNA replication: the E2 transcriptional activator (E2TA), the E2 transcriptional repressor (E2TR) and the E8/E2 transcriptional repressor (E8/E2TR). E2TA is a strong activator of papillomaviral promoters and is required for viral DNA replication. E2TR and E8/E2TR inhibit the activities of E2TA but also possess weak transactivational properties of their own....

  3. Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR, a PadR-Like Repressor

    Morabbi Heravi, Kambiz; Lange, Julian; Watzlawick, Hildegard; Kalinowski, Jörn; Altenbuchner, Josef

    2014-01-01

    Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame reveal...

  4. MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function

    Donzelli, S; Fontemaggi, G; Fazi, F; Di Agostino, S; Padula, F; Biagioni, F; Muti, P; Strano, S; Blandino, G

    2012-01-01

    p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21waf1 transcription. p21waf1 protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance. PMID:22193543

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

    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.

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

    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

  7. Involvement of transcription repressor Snail in the regulation of human telomerase reverse transcriptase (hTERT) by transforming growth factor-β.

    Yoo, Young-Sun; Park, Seoyoung; Gwak, Jungsug; Ju, Bong Gun; Oh, Sangtaek

    2015-09-11

    Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is the primary determinant for telomerase enzyme activity, which has been associated with cellular immortality. Expression of the hTERT gene is regulated by various extracellular (external) stimuli and is aberrantly up-regulated in more than 90% of cancers. Here we show that hTERT gene expression was repressed in response to transforming growth factor-β (TGF-β) by a mechanism dependent on transcription factors Snail and c-Myc. TGF-β activated Snail and down-regulated c-Myc gene expression. In addition, ectopic expression of Snail strongly inhibited hTERT promoter activity, although co-expression of c-Myc abrogated this effect. Chromatin immunoprecipitation (ChIP) analysis revealed that TGF-β decreased c-Myc occupancy and dramatically increased recruitment of Snail to the E-box motifs of the hTERT promoter, thereby repressing hTERT expression. Our findings suggest a dynamic alteration in hTERT promoter occupancy by Snail and c-Myc is the mechanistic basis for TGF-β-mediated regulation of hTERT. PMID:26235880

  8. STENOFOLIA acts as a repressor in regulating leaf blade outgrowth

    Lin, Hao; Niu, Lifang; Tadege, Million

    2013-01-01

    We recently reported that the Medicago WOX gene, STENOFOLIA (STF), acts as a transcriptional repressor in regulating leaf blade outgrowth. By using the Nicotiana sylvestris bladeless lam1 mutant as a genetic tool, we showed that the WUS-box, which is conserved among WUS clade WOX genes, is partly responsible for the repressive activity of STF. All members of the modern/WUS clade genes (WUS, WOX1-WOX7) in Arabidopsis that contain intact WUS-box can substitute for STF/LAM1 function while the in...

  9. Chromatin structure near transcriptionally active genes

    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

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

    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.

  11. Transcriptional Regulation of the AP-2α Promoter by BTEB-1 and AP-2rep, a Novel wt-1/egr-Related Zinc Finger Repressor

    Imhof, Axel; Schuierer, Marion; Werner, Oliver; Moser, Markus; Roth, Christina; Bauer, Reinhard; BUETTNER, REINHARD

    1999-01-01

    AP-2 transcription factors have been suggested to exert key regulatory functions in vertebrate embryonic development, in tumorigenicity of various cancer cell types, and in controlling cell cycle and apoptotic effector genes. In this study, we investigated transcriptional regulation of the AP-2α gene promoter mediated by an autoregulatory element (referred to as A32) with a core consensus AP-2 binding site at position −336 relative to the mRNA initiation site. AP-2 and multiple different nucl...

  12. Novel insights into the regulation of the antioxidant response element mediated gene expression by electrophiles: induction of the transcriptional repressor BACH1 by NRF2

    Jyrkkänen, Henna-Kaisa; Suvi M. Kuosmanen; Heinäniemi, Merja; Laitinen, Heidi; Kansanen, Emilia; Mella-Aho, Eero; Leinonen, Hanna; Ylä-Herttuala, Seppo; Levonen, Anna-Liisa

    2011-01-01

    Abstract A central mechanism in cellular defence against oxidative or electrophilic stress is mediated by transcriptional induction of genes via the Antioxidant Response Element (ARE), a cis-acting sequence present in the regulatory regions of genes involved in the detoxification and elimination of reactive oxidants and electrophiles. The ARE binds different basic-region leucine zipper (bZIP) transcription factors, most notably NF-E2 related factor-2 (Nrf2) that functions as a tran...

  13. Transcription-responsive regulation of c-myc proto-oncogene – structural and biophysical studies

    Cukier, C. D.

    2010-01-01

    The Far-UpStream Element (FUSE) regulatory system tightly controls the expression of c-myc proto-oncogene – a master regulator of cellular proliferation and differentiation. The FUSE mechanism relies on the inter-molecular interactions between a DNA regulatory sequence – the FUSE, a transcriptional activator – FUSEBinding Protein (FBP) and a transcriptional repressor – FBP-Interacting Repressor (FIR). The FUSE DNA element serves as a sensor of the level of ongoing c-myc tran...

  14. Are corepressors always repressors?

    Andrea Hessenauer

    2011-08-01

    Full Text Available In this review we summarize data on paradox actions of corepressors, acting under certain circumstances as activators of transcription. Putative mechanisms, including the role of splice variants, recruitment of coactivators by corepressors and the importance of chromatin structure and hormone response elements are discussed.

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

    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

  16. The mammalian Cut homeodomain protein functions as a cell-cycle-dependent transcriptional repressor which downmodulates p21WAF1/CIP1/SDI1 in S phase.

    Coqueret, O; Bérubé, G; Nepveu, A

    1998-01-01

    Cut is a homeodomain transcription factor which has the unusual property of containing several DNA-binding domains: three regions called Cut repeats and the Cut homeodomain. Genetic studies in Drosophila melanogaster indicate that cut plays important roles in the determination and maintenance of cell-type specificity. In the present study, we show that mammalian Cut proteins may yet play another biological role, specifically in proliferating cells. We found that the binding of Cut to a consen...

  17. GLI3 repressor controls nephron number via regulation of Wnt11 and Ret in ureteric tip cells.

    Jason E Cain

    Full Text Available Truncating GLI3 mutations in Pallister-Hall Syndrome with renal malformation suggests a requirement for Hedgehog signaling during renal development. HH-dependent signaling increases levels of GLI transcriptional activators and decreases processing of GLI3 to a shorter transcriptional repressor. Previously, we showed that Shh-deficiency interrupts early inductive events during renal development in a manner dependent on GLI3 repressor. Here we identify a novel function for GLI3 repressor in controlling nephron number. During renal morphogenesis, HH signaling activity, assayed by expression of Ptc1-lacZ, is localized to ureteric cells of the medulla, but is undetectable in the cortex. Targeted inactivation of Smo, the HH effector, in the ureteric cell lineage causes no detectable abnormality in renal morphogenesis. The functional significance of absent HH signaling activity in cortical ureteric cells was determined by targeted deletion of Ptc1, the SMO inhibitor, in the ureteric cell lineage. Ptc1(-/-UB mice demonstrate ectopic Ptc1-lacZ expression in ureteric branch tips and renal hypoplasia characterized by reduced kidney size and a paucity of mature and intermediate nephrogenic structures. Ureteric tip cells are remarkable for abnormal morphology and impaired expression of Ret and Wnt11, markers of tip cell differentiation. A finding of renal hypoplasia in Gli3(-/- mice suggests a pathogenic role for reduced GLI3 repressor in the Ptc1(-/-UB mice. Indeed, constitutive expression of GLI3 repressor via the Gli3(Delta699 allele in Ptc1(-/-UB mice restores the normal pattern of HH signaling, and expression of Ret and Wnt11 and rescued the renal phenotype. Thus, GLI3 repressor controls nephron number by regulating ureteric tip cell expression of Wnt11 and Ret.

  18. Promoter proximal polyadenylation sites reduce transcription activity

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

    2012-01-01

    transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

  19. The rice GERMINATION DEFECTIVE 1, encoding a B3 domain transcriptional repressor, regulates seed germination and seedling development by integrating GA and carbohydrate metabolism

    Guo, Xiaoli; Hou, Xiaomei; FANG, JUN; Wei, Piwei; Xu, Bo; Chen, Mingluan; Feng, Yuqi; Chu, Chengcai

    2013-01-01

    It has been shown that seed development is regulated by a network of transcription factors in Arabidopsis including LEC1 (LEAFY COTYLEDON1), L1L (LEC1-like) and the B3 domain factors LEC2, FUS3 (FUSCA3) and ABI3 (ABA-INSENSITIVE3); however, molecular and genetic regulation of seed development in cereals is poorly understood. To understand seed development and seed germination in cereals, a large-scale screen was performed using our T–DNA mutant population, and a mutant germination-defective1 ...

  20. Transcription of the uvrD gene of Escherichia coli is controlled by the lexA repressor and by attenuation.

    Easton, A M; Kushner, S R

    1983-01-01

    The nucleotide sequence of the control region and the presumptive N-terminal portion of the uvrD gene of Escherichia coli K-12 has been determined. The 1190 base pairs of DNA examined include the likely coding sequence for the first 258 amino acids of the uvrD protein. The transcription promoter for the uvrD gene was identified upstream of the protein coding region. Synthesis of messenger RNA in vitro from this promoter was inhibited by purified lexA protein. The lexA protein was found to bin...

  1. Human cytomegalovirus IE2 protein interacts with transcription activating factors

    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.

  2. Structural Analysis of Iac Repressor Bound to Allosteric Effectors

    Daber,R.; Stayrook, S.; Rosenberg, A.; Lewis, M.

    2007-01-01

    The lac operon is a model system for understanding how effector molecules regulate transcription and are necessary for allosteric transitions. The crystal structures of the lac repressor bound to inducer and anti-inducer molecules provide a model for how these small molecules can modulate repressor function. The structures of the apo repressor and the repressor bound to effector molecules are compared in atomic detail. All effectors examined here bind to the repressor in the same location and are anchored to the repressor through hydrogen bonds to several hydroxyl groups of the sugar ring. Inducer molecules form a more extensive hydrogen-bonding network compared to anti-inducers and neutral effector molecules. The structures of these effector molecules suggest that the O6 hydroxyl on the galactoside is essential for establishing a water-mediated hydrogen bonding network that bridges the N-terminal and C-terminal sub-domains. The altered hydrogen bonding can account in part for the different structural conformations of the repressor, and is vital for the allosteric transition.

  3. A role of transcriptional activators as antirepressors for the autoinhibitory activity of TATA box binding of transcription factor IID

    Kotani, Tomohiro; Banno, Ken-ichi; Ikura, Mitsuhiko; Hinnebusch, Alan G.; Nakatani, Yoshihiro; Kawaichi, Masashi; Kokubo, Tetsuro

    2000-01-01

    The TATA box-binding activity of transcription factor IID (TFIID) is autoinhibited by the N-terminal domain of the Drosophila TATA box-binding protein- (TBP) associated factor 230/yeast TBP-associated factor 145 subunit, which binds to the TATA box-binding domain of TBP by mimicking the TATA box structure. Here, we propose a mechanism of transcriptional activation that involves antirepression of this autoinhibitory activity by transcriptional activators. Like the autoinhibitory domain of TFII...

  4. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J

    2016-04-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  5. Crystallization and preliminary X-ray diffraction analysis of the TetR-family transcriptional repressor YhgD from Bacillus halodurans

    Triclinic crystals of YhgD from B. halodurans have been obtained. X-ray data have been collected to 1.9 Å resolution using synchrotron radiation. YhgD is a member of the TetR-family transcription factors, which regulate genes encoding proteins involved in multidrug resistance, virulence, osmotic stress and pathogenicity. YhgD from the alkaliphilic bacterium Bacillus halodurans was cloned and overexpressed in Escherichia coli. YhgD (Bh2145) from B. halodurans is composed of 193 amino-acid residues with a molecular mass of 21 853 Da. YhgD was crystallized at 296 K using ethylene glycol as a precipitant by the sitting-drop vapour-diffusion method. The crystal diffracted to 1.9 Å resolution and belonged to the apparent triclinic space group P1, with unit-cell parameters a = 37.22, b = 47.85, c = 54.15 Å, α = 92.75, β = 107.9, γ = 90.27°. The asymmetric unit is likely to contain two molecules of monomeric YhgD, giving a crystal volume per mass (VM) of 2.05 Å3 Da−1 and a solvent content of 40.2%

  6. Nuclear factor I revealed as family of promoter binding transcription activators

    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.

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

    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...

  8. Characterization of human UTF1, a chromatin-associated protein with repressor activity expressed in pluripotent cells

    Kooistra, Susanne M.; Thummer, Rajkumar P.; Eggen, Bart J. L.

    2009-01-01

    In mice, during early embryonic development UTF1 (undifferentiated embryonic cell transcription factor 1) is expressed in the inner cell mass of blastocysts and in adult animals expression is restricted to the gonads. (Embryonic) Cells expressing UTF1 are generally considered pluripotent, meaning th

  9. Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli

    Søgaard-Andersen, Lotte; Møllegaard, N E; Douthwaite, S R; Valentin-Hansen, P

    1990-01-01

    We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor. As is characteristic for CytR-regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP-CRP. One of these sites, CRP-1, overlaps the -35...... region, and is sufficient for activation; the second site, CRP-2, centred around -93, is indispensable for repression. Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP-2, but also on CRP-1 and the length and possibly the sequence separating...... these two sites. Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co-operativity is observed in the interaction of CytR with the two CRP binding sites. Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP-CRP to the mutated CRP...

  10. Dual effects of TGF-β on ERα-mediated estrogenic transcriptional activity in breast cancer

    Cao Xu

    2009-11-01

    Full Text Available Abstract Background TGF-β resistance often develops in breast cancer cells that in turn overproduce this cytokine to create a local immunosuppressive environment that fosters tumor growth and exacerbates the invasive and metastatic behavior of the tumor cells themselves. Smads-mediated cross-talk with the estrogen receptor has been implied to play an important role in development and/or progression of breast cancer. We investigated how TGF-β regulates ERα-induced gene transcription and potential mechanisms of frequent TGF-β resistance in breast cancer. Methods Effect of TGF-β on ERα-mediated gene transcription was investigated in breast cancer cell lines using transient transfection, real-time PCR, sequential DNA precipitation, and small interfering RNA assays. The expression of Smads on both human breast cancer cell lines and ERα-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays. Results A complex of Smad3/4 mediates TGF-β inhibition of ERα-mediated estrogenic activity of gene transcription in breast cancer cells, and Smad4 is essential and sufficient for such repression. Either overexpression of Smad3 or inhibition of Smad4 leads to the "switch" of TGF-β from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERα-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma. Conclusion These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-β suppression of ERα transactivity in breast cancer cells.

  11. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

    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

  12. LSD1 co-repressor Rcor2 orchestrates neurogenesis in the developing mouse brain.

    Wang, Yixuan; Wu, Qian; Yang, Peng; Wang, Chenfei; Liu, Jing; Ding, Wenyu; Liu, Wensu; Bai, Ye; Yang, Yuanyuan; Wang, Hong; Gao, Shaorong; Wang, Xiaoqun

    2016-01-01

    Epigenetic regulatory complexes play key roles in the modulation of transcriptional regulation underlying neural stem cell (NSC) proliferation and progeny specification. How specific cofactors guide histone demethylase LSD1/KDM1A complex to regulate distinct NSC-related gene activation and repression in cortical neurogenesis remains unclear. Here we demonstrate that Rcor2, a co-repressor of LSD1, is mainly expressed in the central nervous system (CNS) and plays a key role in epigenetic regulation of cortical development. Depletion of Rcor2 results in reduced NPC proliferation, neuron population, neocortex thickness and brain size. We find that Rcor2 directly targets Dlx2 and Shh, and represses their expressions in developing neocortex. In addition, inhibition of Shh signals rescues the neurogenesis defects caused by Rcor2 depletion both in vivo and in vitro. Hence, our findings suggest that co-repressor Rcor2 is critical for cortical development by repressing Shh signalling pathway in dorsal telencephalon. PMID:26795843

  13. Characterization of human UTF1, a chromatin-associated protein with repressor activity expressed in pluripotent cells

    Susanne M Kooistra; Thummer, Rajkumar P.; Eggen, Bart J.L.

    2009-01-01

    In mice, during early embryonic development UTF1 (undifferentiated embryonic cell transcription factor 1) is expressed in the inner cell mass of blastocysts and in adult animals expression is restricted to the gonads. (Embryonic) Cells expressing UTF1 are generally considered pluripotent, meaning they can differentiate into all cell types of the adult body. In mouse it was shown that UTF1 is tightly associated with chromatin and that it is required for proper differentiation of embryonic carc...

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

    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

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

    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

  16. Subspecialization of R2R3-MYB Repressors for Anthocyanin and Proanthocyanidin Regulation in Forage Legumes

    Albert, Nick W.

    2015-01-01

    The synthesis of anthocyanin pigments and proanthocyanidins (condensed tannins) is regulated by MYB-bHLH-WDR (MBW) transcription factor complexes in all angiosperms studied to date. Tr-MYB133 and Tr-MYB134 were isolated from Trifolium repens and encode R2R3-MYBs that antagonize the activity of MBW activation complexes. These two genes are conserved in other legume species, and form two sub-clades within the larger anthocyanin/proanthocyanidin clade of MYB repressors. However, unlike petunia a...

  17. The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription.

    Hiebert, S W; Sun, W; Davis, J. N.; Golub, T; Shurtleff, S; Buijs, A; Downing, J R; Grosveld, G; Roussell, M F; Gilliland, D G; Lenny, N; Meyers, S

    1996-01-01

    The t(12;21) translocation is present in up to 30% of childhood B-cell acute lymphoblastic and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1. The t(12;21) translocation encodes a 93-kDa fusion protein that localizes to a high-salt- and detergent-resistant nuclear compartment. This protein binds the enhancer core motif, TGTGGT, and interacts with the AML-1-binding protein, core-binding factor beta. Although TEL/AML-1B retains the C-terminal doma...

  18. The korF region of broad-host-range plasmid RK2 encodes two polypeptides with transcriptional repressor activity.

    Jagura-Burdzy, G; Ibbotson, J P; Thomas, C M

    1991-01-01

    Broad-host-range IncP plasmid RK2 possesses a series of operons involved in plasmid maintenance, whose expression is coordinated by a number of regulators, most of which are encoded in the central regulatory korA-korB operon. The nucleotide sequence of two new cistrons in this operon, comprising what we have previously designated the korF locus located between coordinates 57.0 and 56.0 kb on the genome of the IncP alpha plasmid RK2, is presented. The cistrons encode polypeptides of 173 and 17...

  19. Roles of transcriptional factor 7 in production of inflammatory factors for lung diseases

    Zhu, Yichun; Wang, William; Wang, Xiangdong

    2015-01-01

    Lung disease is the major cause of death and hospitalization worldwide. Transcription factors such as transcription factor 7 (TCF7) are involved in the pathogenesis of lung diseases. TCF7 is important for T cell development and differentiation, embryonic development, or tumorogenesis. Multiple TCF7 isoforms can be characterized by the full-length isoform (FL-TCF7) as a transcription activator, or dominant negative isoform (dn-TCF7) as a transcription repressor. TCF7 interacts with multiple pr...

  20. The retinoblastoma protein as a transcriptional repressor

    Helin, K; Ed, H

    1993-01-01

    The retinoblastoma protein (pRB) is one of the best-studied tumour suppressor gene products. Its loss during the genesis of many human tumours, its inactivation by several DNA tumour virus oncoproteins, and its ability to inhibit cell growth when introduced into dividing cells all suggest that p...

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

    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

  2. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Iñigo; Novick, Richard P; Christie, Gail E; Penadés, José R

    2013-08-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

  3. The transcriptional repressor ZBP-89 and the lack of Sp1/Sp3, c-Jun, and Stat3 are important for the down-regulation of the vimentin gene during C2C12 myogenesis

    Salmon, Morgan; Zehner, Zendra E.

    2009-01-01

    Currently, considerable information is available about how muscle-specific genes are activated during myogenesis, yet little is known about how non-muscle genes are down-regulated. The intermediate filament protein vimentin is known to be “turned off” during myogenesis to be replaced by desmin, the muscle-specific intermediate filament protein. Here, we demonstrate that vimentin down-regulation is the result of the combined effect of several transcription factors. Levels of the positive activ...

  4. The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling1[OPEN

    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

  5. A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing

    Zangger Nadine

    2011-07-01

    Full Text Available Abstract Background KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences. Method To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression. Results We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes. Conclusion Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.

  6. Crystal Structure of the Lactose Operon Repressor and Its Complexes with DNA and Inducer

    Lewis, Mitchell; Chang, Geoffrey; Horton, Nancy C.; Kercher, Michele A.; Pace, Helen C.; Schumacher, Maria A.; Brennan, Richard G.; Lu, Ponzy

    1996-03-01

    The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-β-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

  7. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB.

    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

  8. H-NS is a repressor of major virulence gene loci in Vibrio parahaemolyticus

    Dongsheng eZhou

    2014-12-01

    Full Text Available Vibrio parahaemolyticus, a leading cause of seafood-associated diarrhea and gastroenteritis, harbors three major virulence gene loci T3SS1, Vp-PAI (T3SS1+tdh2 and T6SS2. As showing is this study, the nucleoid-associated DNA-binding regulator H-NS binds to multiple promoter-proximal regions in each of the above three loci to repress their transcription, and moreover H-NS inhibits the cytotoxicitiy, enterotoxicity, hemolytic activity, and mouse lethality of V. parahaemolyticus. H-NS appears to act as a major repressor of the virulence of this pathogen.

  9. BDNF selectively regulates GABAA receptor transcription by activation of the JAK/STAT pathway.

    Lund, Ingrid V; Hu, Yinghui; Raol, YogendraSinh H; Benham, Rebecca S; Faris, Ramona; Russek, Shelley J; Brooks-Kayal, Amy R

    2008-01-01

    The gamma-aminobutyric acid (GABA) type A receptor (GABA(A)R) is the major inhibitory neurotransmitter receptor in the brain. Its multiple subunits show regional, developmental, and disease-related plasticity of expression; however, the regulatory networks controlling GABA(A)R subunit expression remain poorly understood. We report that the seizure-induced decrease in GABA(A)R alpha1 subunit expression associated with epilepsy is mediated by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway regulated by brain-derived neurotrophic factor (BDNF). BDNF- and seizure-dependent phosphorylation of STAT3 cause the adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) family member ICER (inducible cAMP early repressor) to bind with phosphorylated CREB at the Gabra1:CRE site. JAK/STAT pathway inhibition prevents the seizure-induced decrease in GABA(A)R alpha1 abundance in vivo and, given that BDNF is known to increase the abundance of GABA(A)R alpha4 in a JAK/STAT-independent manner, indicates that BDNF acts through at least two distinct pathways to influence GABA(A)R-dependent synaptic inhibition. PMID:18922788

  10. Potential Role of Activating Transcription Factor 5 during Osteogenesis

    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.

  11. Repressors report fewer intrusions following a laboratory stressor : The role of reduced stressor-relevant concept activation and inhibitory functioning

    Overwijk, Sippie; Wessel, Ineke; de Jong, Peter J.

    2009-01-01

    This study investigated whether a repressive coping style is associated with fewer intrusions following an experimentally controlled stressor. Furthermore, we examined whether lower activation of stressor-relevant concepts in long-term memory and better inhibitory functioning may contribute to this

  12. A novel human BTB-kelch protein KLHL31, strongly expressed in muscle and heart, inhibits transcriptional activities of TRE and SRE.

    Yu, Weishi; Li, Yongqing; Zhou, Xijin; Deng, Yun; Wang, Zequn; Yuan, Wuzhou; Li, Dali; Zhu, Chuanbing; Zhao, Xueying; Mo, Xiaoyang; Huang, Wen; Luo, Na; Yan, Yan; Ocorr, Karen; Bodmer, Rolf; Wang, Yuequn; Wu, Xiushan

    2008-11-30

    The Bric-a-brac, Tramtrack, Broad-complex (BTB) domain is a protein-protein interaction domain that is found in many zinc finger transcription factors. BTB containing proteins play important roles in a variety of cellular functions including regulation of transcription, regulation of the cytoskeleton, protein ubiquitination, angiogenesis, and apoptosis. Here, we report the cloning and characterization of a novel human gene, KLHL31, from a human embryonic heart cDNA library. The cDNA of KLHL31 is 5743 bp long, encoding a protein product of 634 amino acids containing a BTB domain. The protein is highly conserved across different species. Western blot analysis indicates that the KLHL31 protein is abundantly expressed in both embryonic skeletal and heart tissue. In COS-7 cells, KLHL31 proteins are localized to both the nucleus and the cytoplasm. In primary cultures of nascent mouse cardiomyocytes, the majority of endogenous KLHL31 proteins are localized to the cytoplasm. KLHL31 acts as a transcription repressor when fused to GAL4 DNA-binding domain and deletion analysis indicates that the BTB domain is the main region responsible for this repression. Overexpression of KLHL31 in COS-7 cells inhibits the transcriptional activities of both the TPA-response element (TRE) and serum response element (SRE). KLHL31 also significantly reduces JNK activation leading to decreased phosphorylation and protein levels of the JNK target c-Jun in both COS-7 and Hela cells. These results suggest that KLHL31 protein may act as a new transcriptional repressor in MAPK/JNK signaling pathway to regulate cellular functions. PMID:18719355

  13. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    Søgaard-Andersen, Lotte; Martinussen, J; Møllegaard, N E; Douthwaite, S R; Valentin-Hansen, P

    1990-01-01

    We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) Cyt...

  14. First Exon Length Controls Active Chromatin Signatures and Transcription

    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.

  15. Centromeric Transcription Regulates Aurora-B Localization and Activation

    Michael D. Blower

    2016-05-01

    Full Text Available Centromeric transcription is widely conserved; however, it is not clear what role centromere transcription plays during mitosis. Here, I find that centromeres are transcribed in Xenopus egg extracts into a long noncoding RNA (lncRNA; cen-RNA that localizes to mitotic centromeres, chromatin, and spindles. cen-RNAs bind to the chromosomal passenger complex (CPC in vitro and in vivo. Blocking transcription or antisense inhibition of cen-RNA leads to a reduction of CPC localization to the inner centromere and misregulation of CPC component Aurora-B activation independently of known centromere recruitment pathways. Additionally, transcription is required for normal bipolar attachment of kinetochores to the mitotic spindle, consistent with a role for cen-RNA in CPC regulation. This work demonstrates that cen-RNAs promote normal kinetochore function through regulation of the localization and activation of the CPC and confirm that lncRNAs are components of the centromere.

  16. Isolated HIV-1 core is active for reverse transcription

    Harrich David; Stenzel Deborah; Warrilow David

    2007-01-01

    Abstract Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles...

  17. Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation.

    Papadopoulou, Thaleia; Kaymak, Aysegül; Sayols, Sergi; Richly, Holger

    2016-06-01

    Mediator is considered an enhancer of RNA-Polymerase II dependent transcription but its function and regulation in pluripotent mouse embryonic stem cells (mESCs) remains unresolved. One means of controlling the function of Mediator is provided by the binding of the Cdk8 module (Med12, Cdk8, Ccnc and Med13) to the core Mediator. Here we report that Med12 operates together with PRC1 to silence key developmental genes in pluripotency. At the molecular level, while PRC1 represses genes it is also required to assemble ncRNA containing Med12-Mediator complexes. In the course of cellular differentiation the H2A ubiquitin binding protein Zrf1 abrogates PRC1-Med12 binding and facilitates the association of Cdk8 with Mediator. This remodeling of Mediator-associated protein complexes converts Mediator from a transcriptional repressor to a transcriptional enhancer, which then mediates ncRNA-dependent activation of Polycomb target genes. Altogether, our data reveal how the interplay of PRC1, ncRNA and Mediator complexes controls pluripotency and cellular differentiation. PMID:27096886

  18. Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro.

    Hammerl, Jens Andre; Jäckel, Claudia; Lanka, Erich; Roschanski, Nicole; Hertwig, Stefan

    2016-01-01

    Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages. PMID:27527206

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

    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

  20. Cooperative activation of transcription by autoimmune regulator AIRE and CBP

    Autoimmune regulator (AIRE) is a transcriptional regulator that is believed to control the expression of tissue-specific genes in the thymus. Mutated AIRE is responsible for onset of the hereditary autoimmune disease APECED. AIRE is able to form nuclear bodies (NBs) and interacts with the ubiquitous transcriptional coactivator CBP. In this paper, we show that CBP and AIRE synergistically activate transcription on different promoter reporters whereas AIRE gene mutation R257X, found in APECED patients, interferes with this coactivation effect. Furthermore, the overexpression of AIRE and CBP collaboratively enhance endogenous IFNβ mRNA expression. The immunohistochemical studies suggest that CBP, depending on the balance of nuclear proteins, is a component of AIRE NBs. We also show that AIRE NBs are devoid of active chromatin and, therefore, not sites of transcription. In addition, we demonstrate by 3D analyses that AIRE and CBP, when colocalizing, are located spatially differently within AIRE NBs. In conclusion, our data suggest that AIRE activates transcription of the target genes, i.e., autoantigens in collaboration with CBP and that this activation occurs outside of AIRE NBs

  1. Estrogen directly activates AID transcription and function

    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...

  2. Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity

    Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis

  3. Trans-dominant inhibition of transcription activator LFB1.

    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 ...

  4. PKG-1α mediates GATA4 transcriptional activity.

    Ma, Yanlin; Wang, Jun; Yu, Yanhong; Schwartz, Robert J

    2016-06-01

    GATA4, a zinc-finger transcription factor, is central for cardiac development and diseases. Here we show that GATA4 transcriptional activity is mediated by cell signaling via cGMP dependent PKG-1α activity. Protein kinase G (PKG), a serine/tyrosine specific kinase is the major effector of cGMP signaling. We observed enhanced transcriptional activity elicited by co-expressed GATA4 and PKG-1α. Phosphorylation of GATA4 by PKG-1α was detected on serine 261 (S261), while the C-terminal activation domain of GATA4 associated with PKG-1α. GATA4's DNA binding activity was enhanced by PKG-1α via by both phosphorylation and physical association. More importantly, a number of human disease-linked GATA4 mutants exhibited impaired S261 phosphorylation, pointing to defective S261 phosphorylation in the elaboration of human heart diseases. We showed S261 phosphorylation was favored by PKG-1α but not by PKA, and several other kinase signaling pathways such as MAPK and PKC. Our observations demonstrate that cGMP-PKG signaling mediates transcriptional activity of GATA4 and links defective GATA4 and PKG-1α mutations to the development of human heart disease. PMID:26946174

  5. Identification of Quaternary Structure and Functional Domains of the CI Repressor from Bacteriophage TP901-1

    Pedersen, Margit; Lo Leggio, Leila; Grossmann, J. Günter;

    2008-01-01

    The bacteriophage-encoded repressor protein plays a key role in determining the life cycle of a temperate phage following infection of a sensitive host. The repressor protein Cl, which is encoded by the temperate lactococcal phage TP901-1, represses transcription from both the lytic promoter P...... the protein is involved in the interaction with host proteins. By using small-angle X-ray scattering, we show for the first time the overall solution structure of a full-length wild-type bacteriophage repressor at low resolution revealing that the TP901-1 repressor forms a flat oligomer, most probably...

  6. Bacteriophage 434 Hex Protein Prevents RecA-Mediated Repressor Autocleavage

    Paul Shkilnyj

    2013-01-01

    Full Text Available In a λimm434 lysogen, two proteins are expressed from the integrated prophage. Both are encoded by the same mRNA whose transcription initiates at the PRM promoter. One protein is the 434 repressor, needed for the establishment and maintenance of lysogeny. The other is Hex which is translated from an open reading frame that apparently partially overlaps the 434 repressor coding region. In the wild type host, disruption of the gene encoding Hex destabilizes λimm434 lysogens. However, the hex mutation has no effect on lysogen stability in a recA− host. These observations suggest that Hex functions by modulating the ability of RecA to stimulate 434 repressor autocleavage. We tested this hypothesis by identifying and purifying Hex to determine if this protein inhibited RecA‑stimulated autocleavage of 434 repressor in vitro. Our results show that in vitro a fragment of Hex prevents RecA-stimulated autocleavage of 434 repressor, as well as the repressors of the closely related phage P22. Surprisingly, Hex does not prevent RecA‑stimulated autocleavage of phage lambda repressor, nor the E. coli LexA repressor.

  7. TBP domain symmetry in basal and activated archaeal transcription.

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

    2009-01-01

    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 the Methanococcales and Thermoplasmatales, including complete conservation of their N- and C-terminal stirrups; along with helix H'1, the C-terminal stirrup of TBP forms the main interface with TFB/TFIIB. Here, we show that, in stark contrast to its eukaryotic counterparts, multiple substitutions in the C-terminal stirrup of Methanocaldococcus jannaschii (Mja) TBP do not completely abrogate basal transcription. Using DNA affinity cleavage, we show that, by assembling TFB through its conserved N-terminal stirrup, Mja TBP is in effect ambidextrous with regard to basal transcription. In contrast, substitutions in either its N- or the C-terminal stirrup abrogate activated transcription in response to the Lrp-family transcriptional activator Ptr2. PMID:19007415

  8. Metalloregulator CueR biases RNA polymerase's kinetic sampling of dead-end or open complex to repress or activate transcription.

    Martell, Danya J; Joshi, Chandra P; Gaballa, Ahmed; Santiago, Ace George; Chen, Tai-Yen; Jung, Won; Helmann, John D; Chen, Peng

    2015-11-01

    Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription. PMID:26483469

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

    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.

  10. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

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

    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...

  12. Transcriptional stochasticity in gene expression.

    Lipniacki, Tomasz; Paszek, Pawel; Marciniak-Czochra, Anna; Brasier, Allan R; Kimmel, Marek

    2006-01-21

    Due to the small number of copies of molecular species involved, such as DNA, mRNA and regulatory proteins, gene expression is a stochastic phenomenon. In eukaryotic cells, the stochastic effects primarily originate in regulation of gene activity. Transcription can be initiated by a single transcription factor binding to a specific regulatory site in the target gene. Stochasticity of transcription factor binding and dissociation is then amplified by transcription and translation, since target gene activation results in a burst of mRNA molecules, and each mRNA copy serves as a template for translating numerous protein molecules. In the present paper, we explore a mathematical approach to stochastic modeling. In this approach, the ordinary differential equations with a stochastic component for mRNA and protein levels in a single cells yield a system of first-order partial differential equations (PDEs) for two-dimensional probability density functions (pdf). We consider the following examples: Regulation of a single auto-repressing gene, and regulation of a system of two mutual repressors and of an activator-repressor system. The resulting PDEs are approximated by a system of many ordinary equations, which are then numerically solved. PMID:16039671

  13. Activating transcription factor 4 regulates osteoclast differentiation in mice

    Cao, Huiling; Yu, Shibing; Yao, Zhi; Galson, Deborah L; Jiang, Yu; Zhang, Xiaoyan; Fan, Jie; Lu, Binfeng; Guan, Youfei; Luo, Min; Lai, Yumei; Zhu, Yibei; Kurihara, Noriyoshi; Patrene, Kenneth; Roodman, G. David

    2010-01-01

    Activating transcription factor 4 (ATF4) is a critical transcription factor for osteoblast (OBL) function and bone formation; however, a direct role in osteoclasts (OCLs) has not been established. Here, we targeted expression of ATF4 to the OCL lineage using the Trap promoter or through deletion of Atf4 in mice. OCL differentiation was drastically decreased in Atf4–/– bone marrow monocyte (BMM) cultures and bones. Coculture of Atf4–/– BMMs with WT OBLs or a high concentration of RANKL failed ...

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

    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...

  15. The Positive Transcription Elongation Factor b Is an Essential Cofactor for the Activation of Transcription by Myocyte Enhancer Factor 2

    Nojima, Masanori; Huang, Yehong; Tyagi, Mudit; Kao, Hung-Ying; Fujinaga, Koh

    2008-01-01

    The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyt...

  16. Transcriptional Regulatory Circuits Controlling Brown Fat Development and Activation

    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...

  17. Zooming in on Transcription Preinitiation.

    Gupta, Kapil; Sari-Ak, Duygu; Haffke, Matthias; Trowitzsch, Simon; Berger, Imre

    2016-06-19

    Class II gene transcription commences with the assembly of the Preinitiation Complex (PIC) from a plethora of proteins and protein assemblies in the nucleus, including the General Transcription Factors (GTFs), RNA polymerase II (RNA pol II), co-activators, co-repressors, and more. TFIID, a megadalton-sized multiprotein complex comprising 20 subunits, is among the first GTFs to bind the core promoter. TFIID assists in nucleating PIC formation, completed by binding of further factors in a highly regulated stepwise fashion. Recent results indicate that TFIID itself is built from distinct preformed submodules, which reside in the nucleus but also in the cytosol of cells. Here, we highlight recent insights in transcription factor assembly and the regulation of transcription preinitiation. PMID:27067110

  18. Proto-oncogene FBI-1 represses transcription of p21CIP1 by inhibition of transcription activation by p53 and Sp1.

    Choi, Won-Il; Jeon, Bu-Nam; Yun, Chae-Ok; Kim, Pyung-Hwan; Kim, Sung-Eun; Choi, Kang-Yell; Kim, Se Hoon; Hur, Man-Wook

    2009-05-01

    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 transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1-3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target. PMID:19244234

  19. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. PMID:26598443

  20. Loss of the repressor REST in uterine fibroids promotes aberrant G protein-coupled receptor 10 expression and activates mammalian target of rapamycin pathway

    Varghese, Binny V.; Koohestani, Faezeh; McWilliams, Michelle; Colvin, Arlene; Gunewardena, Sumedha; Kinsey, William H.; Nowak, Romana A.; Nothnick, Warren B.; Chennathukuzhi, Vargheese M.

    2013-01-01

    Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductive-aged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase–protein kinase B/AKT–mammalian target of rapamycin (PI3K/AKT–mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K–AKT–mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids. PMID:23284171

  1. Isolated HIV-1 core is active for reverse transcription

    Harrich David

    2007-10-01

    Full Text Available Abstract Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity.

  2. Isolated HIV-1 core is active for reverse transcription.

    Warrilow, David; Stenzel, Deborah; Harrich, David

    2007-01-01

    Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity. PMID:17956635

  3. Structure/function analysis of the Pantoea stewartii quorum-sensing regulator EsaR as an activator of transcription.

    Schu, Daniel J; Carlier, Aurelien L; Jamison, Katherine P; von Bodman, Susanne; Stevens, Ann M

    2009-12-01

    In Pantoea stewartii subsp. stewartii, two regulatory proteins are key to the process of cell-cell communication known as quorum sensing: the LuxI and LuxR homologues EsaI and EsaR. Most LuxR homologues function as activators of transcription in the presence of their cognate acylated homoserine lactone (AHL) signal. However, EsaR was initially found to function as a repressor in the absence of AHL. Previous studies demonstrated that, in the absence of AHL, EsaR retains the ability to function as a weak activator of the lux operon in recombinant Escherichia coli. Here it is shown that both the N-terminal and the C-terminal domains of EsaR are necessary for positive regulation. A site-directed mutagenesis study, guided by homology modeling to LuxR and TraR, has revealed three critical residues in EsaR that are involved in activation of RNA polymerase. In addition, a native EsaR-activated promoter has been identified, which controls expression of a putative regulatory sRNA in P. stewartii. PMID:19820098

  4. Casein Kinase II Phosphorylation of the Yeast Phospholipid Synthesis Transcription Factor Opi1p*

    Chang, Yu-Fang; Carman, George M.

    2006-01-01

    The transcription factor Opi1p regulates phospholipid synthesis in the yeast Saccharomyces cerevisiae by repressing the expression of several UASINO-containing genes (e.g., INO1). Opi1p repressor activity is most active in inositol-supplemented cells. Regulation of Opi1p repressor activity is mediated by multiple phosphorylations catalyzed by protein kinases A and C. In this work, we showed that Opi1p was also phosphorylated by casein kinase II. Using purified maltose binding protein (MBP)-Op...

  5. Balancing intestinal and systemic inflammation through cell type-specific expression of the aryl hydrocarbon receptor repressor

    Olga Brandstätter; Oliver Schanz; Julia Vorac; Jessica König; Tetsushi Mori; Toru Maruyama; Markus Korkowski; Thomas Haarmann-Stemmann; Dorthe von Smolinski; Schultze, Joachim L.; Josef Abel; Charlotte Esser; Haruko Takeyama; Heike Weighardt; Irmgard Förster

    2016-01-01

    As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the a...

  6. Binding of the unorthodox transcription activator, Crl, to the components of the transcription machinery.

    England, Patrick; Westblade, Lars F; Karimova, Gouzel; Robbe-Saule, Véronique; Norel, Françoise; Kolb, Annie

    2008-11-28

    The small regulatory protein Crl binds to sigmaS, the RNA polymerase stationary phase sigma factor. Crl facilitates the formation of the sigmaS-associated holoenzyme (EsigmaS) and thereby activates sigmaS-dependent genes. Using a real time surface plasmon resonance biosensor, we characterized in greater detail the specificity and mode of action of Crl. Crl specifically forms a 1:1 complex with sigmaS, which results in an increase of the association rate of sigmaS to core RNA polymerase without any effect on the dissociation rate of EsigmaS. Crl is also able to associate with preformed EsigmaS with a higher affinity than with sigmaS alone. Furthermore, even at saturating sigmaS concentrations, Crl significantly increases EsigmaS association with the katN promoter and the productive isomerization of the EsigmaS-katN complex, supporting a direct role of Crl in transcription initiation. Finally, we show that Crl does not bind to sigma70 itself but is able at high concentrations to form a weak and transient 1:1 complex with both core RNA polymerase and the sigma70-associated holoenzyme, leaving open the possibility that Crl might also exert a side regulatory role in the transcriptional activity of additional non-sigmaS holoenzymes. PMID:18818199

  7. Post-translational regulation of Oct4 transcriptional activity.

    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.

  8. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

    Piatek, Agnieszka

    2014-11-14

    Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

  9. The Corynebacterium glutamicum aconitase repressor: scratching around for crystals

    Crystallization of AcnR, a repressor of the aconitase gene in Corynebacterium glutamicum, is reported. Intentional manual scratching of the crystallization plates was applied to induce heterogeneous nucleation. Imperfections on the surfaces of crystallization containers are known to influence crystal formation and are thought to do so by helping to overcome the nucleation barrier. The intentional creation of imperfections has been widely applied to induce crystallization of small molecules, but has not been reported for protein crystallization. Here, the crystallization and preliminary X-ray analysis of the TetR-type aconitase repressor are reported. This regulator was the first transcription factor to be identified in the regulation of the tricarboxylic acid cycle in Corynebacterium glutamicum, an organism that is of special industrial interest and is an emerging model organism for Corynebacterineae. Successful crystallization involved introducing manual scratches on the surface of standard commercial plates, which led to a substantial improvement in crystal nucleation and quality

  10. Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors*S⃞

    Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

    2008-01-01

    FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at ...

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

    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

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

    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

  13. Regulating the regulators: modulators of transcription factor activity.

    Everett, Logan; Hansen, Matthew; Hannenhalli, Sridhar

    2010-01-01

    Gene transcription is largely regulated by DNA-binding transcription factors (TFs). However, the TF activity itself is modulated via, among other things, post-translational modifications (PTMs) by specific modification enzymes in response to cellular stimuli. TF-PTMs thus serve as "molecular switchboards" that map upstream signaling events to the downstream transcriptional events. An important long-term goal is to obtain a genome-wide map of "regulatory triplets" consisting of a TF, target gene, and a modulator gene that specifically modulates the regulation of the target gene by the TF. A variety of genome-wide data sets can be exploited by computational methods to obtain a rough map of regulatory triplets, which can guide directed experiments. However, a prerequisite to developing such computational tools is a systematic catalog of known instances of regulatory triplets. We first describe PTM-Switchboard, a recent database that stores triplets of genes such that the ability of one gene (the TF) to regulate a target gene is dependent on one or more PTMs catalyzed by a third gene, the modifying enzyme. We also review current computational approaches to infer regulatory triplets from genome-wide data sets and conclude with a discussion of potential future research. PTM-Switchboard is accessible at http://cagr.pcbi.upenn.edu/PTMswitchboard / PMID:20827600

  14. Computational Characterization of Modes of Transcriptional Regulation of Nuclear Receptor Genes

    Sharma, Yogita; Chilamakuri, Chandra Sekhar Reddy; Bakke, Marit; Lenhard, Boris

    2014-01-01

    Background: Nuclear receptors are a large structural class of transcription factors that act with their co-regulators and repressors to maintain a variety of biological and physiological processes such as metabolism, development and reproduction. They are activated through the binding of small ligands, which can be replaced by drug molecules, making nuclear receptors promising drug targets. Transcriptional regulation of the genes that encode them is central to gaining a deeper understanding o...

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

    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.

  16. Transcriptional activation domains stimulate initiation and elongation at different times and via different residues.

    Brown, S. A.; Weirich, C S; Newton, E M; Kingston, R E

    1998-01-01

    Transcriptional activators can stimulate multiple steps in the transcription process. We have used GAL4 fusion proteins to characterize the ability of different transcriptional activation domains to stimulate transcriptional elongation on the hsp70 gene in vitro. Stimulation of elongation apparently occurs via a mechanistic pathway different from that of stimulation of initiation: the herpes simplex virus VP16, heat shock factor 1 (HSF1) and amphipathic helix (AH) activation domains all stimu...

  17. Environmental phthalate monoesters activate pregnane X receptor-mediated transcription

    Phthalate esters, widely used as plasticizers in the manufacture of products made of polyvinyl chloride, induce reproductive and developmental toxicities in rodents. The mechanism that underlies these effects of phthalate exposure, including the potential role of members of the nuclear receptor superfamily, is not known. The present study investigates the effects of phthalates on the pregnane X receptor (PXR), which mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The ability of phthalate monoesters to activate PXR-mediated transcription was assayed in a HepG2 cell reporter assay following transfection with mouse PXR (mPXR), human PXR (hPXR), or the hPXR allelic variants V140M, D163G, and A370T. Mono-2-ethylhexyl phthalate (MEHP) increased the transcriptional activity of both mPXR and hPXR (5- and 15-fold, respectively) with EC50 values of 7-8 μM. mPXR and hPXR were also activated by monobenzyl phthalate (MBzP, up to 5- to 6-fold) but were unresponsive to monomethyl phthalate and mono-n-butyl phthalate (M(n)BP) at the highest concentrations tested (300 μM). hPXR-V140M and hPXR-A370T exhibited patterns of phthalate responses similar to the wild-type receptor. By contrast, hPXR-D163G was unresponsive to all phthalate monoesters tested. Further studies revealed that hPXR-D163G did respond to rifampicin, but required approximately 40-fold higher concentrations than wild-type receptor, suggesting that the ligand-binding domain D163G variant has impaired ligand-binding activity. The responsiveness of PXR to activation by phthalate monoesters demonstrated here suggests that these ubiquitous environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals

  18. The bovine papillomavirus type 1 E2 transactivator and repressor proteins use different nuclear localization signals.

    Skiadopoulos, M H; McBride, A A

    1996-02-01

    The E2 gene of bovine papillomavirus type 1 encodes at least three nuclear phosphoproteins that regulate viral transcription and DNA replication. All three proteins have a common C-terminal domain that has DNA-binding and dimerization activities. A basic region in this domain forms an alpha helix which makes direct contact with the DNA target. In this study, it is shown that in addition to its role in DNA binding, this basic region functions as a nuclear localization signal both in the E2 DNA-binding domain and in a heterologous protein. Deletion of this signal sequence resulted in increased accumulation of the E2 transactivator and repressor proteins in the cytoplasm, but nuclear localization was not eliminated. In the full-length transactivator protein, another signal, present in the N-terminal transactivation domain, is used for transport to the nucleus, and the C-terminal nuclear localization signal(s) are masked. The use of different nuclear localization signals could potentially allow differential regulation of the subcellular localization of the E2 transactivator and repressor proteins at some stage in the viral life cycle. PMID:8551571

  19. Cif is negatively regulated by the TetR family repressor CifR.

    MacEachran, Daniel P; Stanton, Bruce A; O'Toole, George A

    2008-07-01

    We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene. PMID:18458065

  20. P/CAF-mediated acetylation regulates the function of the basic helix–loop–helix transcription factor TAL1/SCL

    Huang, Suming; Qiu, Yi; Shi, Yubin; Xu, Zhixiong; Brandt, Stephen J.

    2000-01-01

    The basic helix–loop–helix transcription factor TAL1 (or SCL) is a critical regulator of hematopoietic and vascular development and is misexpressed in the majority of patients with T-cell acute lymphoblastic leukemia. We found previously that TAL1 could interact with transcriptional co-activator and co-repressor complexes possessing histone acetyltransferase and deacetylase activities, respectively. Here, we report that TAL1 is subject to acetylation in vivo and can be acetylated by p300 and ...

  1. Aurora kinase B activity is modulated by thyroid hormone during transcriptional activation of pituitary genes

    Tardáguila, Manuel; González-Gugel, Elena; Sánchez-Pacheco, Aurora

    2011-01-01

    Covalent histone modifications clearly play an essential role in ligand-dependent transcriptional regulation by nuclear receptors. One of the predominant mechanisms used by nuclear receptors to activate or repress target-gene transcription is the recruitment of coregulatory factors capable of covalently modify the amino terminal ends of histones. Here we show that the thyroid hormone (T3) produces a rapid increase in histone H3Ser10 phosphorylation (H3Ser10ph) concomitant to the rapid displac...

  2. bHLH003, bHLH013 and bHLH017 are new targets of JAZ repressors negatively regulating JA responses.

    Sandra Fonseca

    Full Text Available Cell reprogramming in response to jasmonates requires a tight control of transcription that is achieved by the activity of JA-related transcription factors (TFs. Among them, MYC2, MYC3 and MYC4 have been described as activators of JA responses. Here we characterized the function of bHLH003, bHLH013 and bHLH017 that conform a phylogenetic clade closely related to MYC2, MYC3 and MYC4. We found that these bHLHs form homo- and heterodimers and also interact with JAZ repressors in vitro and in vivo. Phenotypic analysis of JA-regulated processes, including root and rosette growth, anthocyanin accumulation, chlorophyll loss and resistance to Pseudomonas syringae, on mutants and overexpression lines, suggested that these bHLHs are repressors of JA responses. bHLH003, bHLH013 and bHLH017 are mainly nuclear proteins and bind DNA with similar specificity to that of MYC2, MYC3 and MYC4, but lack a conserved activation domain, suggesting that repression is achieved by competition for the same cis-regulatory elements. Moreover, expression of bHLH017 is induced by JA and depends on MYC2, suggesting a negative feed-back regulation of the activity of positive JA-related TFs. Our results suggest that the competition between positive and negative TFs determines the output of JA-dependent transcriptional activation.

  3. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

    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

  4. H-NS mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO

    Westra, Edze Rients; Pul, Ümit; Heidrich, Nadja; Jore, Matthijs Miklas; Lundgren, Magnus; Stratmann, Thomas; Wurm, Reinhild; Raine, Amanda; Mescher, Melina; Heereveld, Luc Van; Mastop, Marieke; Wagner, E. Gerhart H.; Schnetz, Karin; van der Oost, John; Wagner, Rolf

    2010-01-01

    Abstract The recently discovered prokaryotic CRISPR/Cas defense system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array, is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-vira...

  5. Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2

    Wu, Di; Zhu, Xiaoxi; Jimenez-Cowell, Kevin; Mold, Alexander J.; Sollecito, Christopher C.; Lombana, Nicholas; Jiao, Meng; Wei, Qize

    2015-01-01

    Pitx2 is a bicoid-related homeobox transcription factor implicated in regulating left-right patterning and organogenesis. However, only a limited number of Pitx2 downstream target genes have been identified and characterized. Here we demonstrate that Pitx2 is a transcriptional repressor of DEP domain containing 1B (DEPDC1B). The first intron of the human and mouse DEP domain containing 1B genes contains multiple consensus DNA-binding sites for Pitx2. Chromatin immunoprecipitation assays revea...

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

    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...

  7. Role of co-regulators in metabolic and transcriptional actions of thyroid hormone.

    Astapova, Inna

    2016-04-01

    Thyroid hormone (TH) controls a wide range of physiological processes through TH receptor (TR) isoforms. Classically, TRs are proposed to function as tri-iodothyronine (T3)-dependent transcription factors: on positively regulated target genes, unliganded TRs mediate transcriptional repression through recruitment of co-repressor complexes, while T3 binding leads to dismissal of co-repressors and recruitment of co-activators to activate transcription. Co-repressors and co-activators were proposed to play opposite roles in the regulation of negative T3 target genes and hypothalamic-pituitary-thyroid axis, but exact mechanisms of the negative regulation by TH have remained elusive. Important insights into the roles of co-repressors and co-activators in different physiological processes have been obtained using animal models with disrupted co-regulator function. At the same time, recent studies interrogating genome-wide TR binding have generated compelling new data regarding effects of T3, local chromatin structure, and specific response element configuration on TR recruitment and function leading to the proposal of new models of transcriptional regulation by TRs. This review discusses data obtained in various mouse models with manipulated function of nuclear receptor co-repressor (NCoR or NCOR1) and silencing mediator of retinoic acid receptor and thyroid hormone receptor (SMRT or NCOR2), and family of steroid receptor co-activators (SRCs also known as NCOAs) in the context of TH action, as well as insights into the function of co-regulators that may emerge from the genome-wide TR recruitment analysis. PMID:26673411

  8. β-Catenin-independent activation of TCF1/LEF1 in human hematopoietic tumor cells through interaction with ATF2 transcription factors.

    Grumolato, Luca; Liu, Guizhong; Haremaki, Tomomi; Mungamuri, Sathish Kumar; Mong, Phyllus; Akiri, Gal; Lopez-Bergami, Pablo; Arita, Adriana; Anouar, Youssef; Mlodzik, Marek; Ronai, Ze'ev A; Brody, Joshua; Weinstein, Daniel C; Aaronson, Stuart A

    2013-01-01

    The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of

  9. Transcriptional and nontranscriptional regulation of NIS activity and radioiodide transport

    Jung, Kyung Ho; Lee, Kyung Han [Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2007-10-15

    Radioiodide transport has been extensively and successfully used in the evaluation and management of thyroid disease. The molecular characterization of the sodium/iodide symporter (NIS) and cloning of the NIS gene has led to the recent expansion of the use of radioiodide to cancers of the breast and other nonthyroidal tissues exogenously transduced with the NIS gene. More recently, discoveries regarding the functional analysis and regulatory processes of the NIS molecule are opening up exciting opportunities for new research and applications for NIS and radioiodide. The success of NIS based cancer therapy is dependent on achievement of maximal radioiodide transport sufficient to allow delivery of effective radiation doses. This in turn relies on high transcription rates of the NIS gene. However, newer discoveries indicate that nontranscriptional processes that regulate NIS trafficking to cell membrane are also critical determinants of radioiodide uptake. In this review, molecular mechanisms that underlie regulation of NIS transcription and stimuli that augment membrane trafficking and functional activation of NIS molecules will be discussed. A better understanding of how the expression and cell surface targeting of NIS proteins is controlled will hopefully aid in optimizing NIS gene based cancer treatment as well as NIS based reporter-gene imaging strategies.

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

    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

  11. Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors

    Id-1, a member of the helix-loop-helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors

  12. Repression of RNA Polymerase I Transcription by the Tumor Suppressor p53

    Zhai, Weiguo; Comai, Lucio

    2000-01-01

    The tumor suppressor protein p53 is frequently inactivated in tumors. It functions as a transcriptional activator as well as a repressor for a number of viral and cellular promoters transcribed by RNA polymerase II (Pol II) and by RNA Pol III. Moreover, it appears that p53 also suppresses RNA Pol I transcription. In this study, we examined the molecular mechanism of Pol I transcriptional inhibition by p53. We show that wild-type, but not mutant, p53 can repress Pol I transcription from a huma...

  13. Chemical modification of arginine residues in the lactose repressor

    The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal. Arginine reaction was quantitated by either amino aced analysis or incorporation of 14C-labeled phenylglyoxal. Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees. The correlation of the decrease in DNA binding activities with the modification of ∼ 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s). For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished. This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions. These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA

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

    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.

  15. The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

    Jens Andre Hammerl

    2015-06-01

    Full Text Available Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\\(_{\\rm{R}}\\3 in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\\(_{\\rm{R}}\\3 upstream of the prophage repressor gene. The O\\(_{\\rm{R}}\\3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages.

  16. The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site.

    Hammerl, Jens Andre; Roschanski, Nicole; Lurz, Rudi; Johne, Reimar; Lanka, Erich; Hertwig, Stefan

    2015-06-01

    Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O\\(_{\\rm{R}}\\)3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O\\(_{\\rm{R}}\\)3) upstream of the prophage repressor gene. The O\\(_{\\rm{R}}\\)3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages. PMID:26043380

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

    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

  18. A single mutation in the core domain of the lac repressor reduces leakiness

    Gatti-Lafranconi, Pietro; Dijkman, Willem; Devenish, Sean RA; Hollfelder, Florian

    2013-01-01

    The lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements

  19. Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells.

    Ikeda, K.; Shaw-White, J R; Wert, S E; Whitsett, J A

    1996-01-01

    Thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3alpha (HNF-3alpha), and HNF-3beta regulate the transcription of genes expressed in the respiratory epithelium. To test whether members of the HNF-3/forkhead family influence TTF-1 gene expression, deletion constructs containing the 5' region of the human TTF-1 gene were transfected into immortalized mouse lung epithelial (MLE) cells. DNase I protection and electrophoretic mobility shift assays identified elements in the 5' reg...

  20. Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells.

    Michael P Storm

    Full Text Available The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2.

  1. Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression.

    Achatz, G; Hölzl, B; Speckmayer, R; Hauser, C; Sandhofer, F; Paulweber, B.

    1997-01-01

    The orphan receptor ARP-1/COUP-TFII, a member of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of nuclear receptors, strongly represses transcriptional activity of numerous genes, including several apolipoprotein-encoding genes. Recently it has been demonstrated that the mechanism by which COUP-TFs reduce transcriptional activity involves active repression and transrepression. To map the domains of ARP-1/COUP-TFII required for repressor activity, a detailed ...

  2. The homeobox genes vox and vent are redundant repressors of dorsal fates in zebrafish.

    Imai, Y; Gates, M A; Melby, A E; Kimelman, D; Schier, A F; Talbot, W S

    2001-06-01

    Ventralizing transcriptional repressors in the Vox/Vent family have been proposed to be important regulators of dorsoventral patterning in the early embryo. While the zebrafish genes vox (vega1) and vent (vega2) both have ventralizing activity in overexpression assays, loss-of-function studies are needed to determine whether these genes have distinct or redundant functions in dorsoventral patterning and to provide critical tests of the proposed regulatory interactions among vox, vent and other genes that act to establish the dorsoventral axis. We show that vox and vent are redundant repressors of dorsal fates in zebrafish. Mutants that lack vox function have little or no dorsoventral patterning defect, and inactivation of either vox or vent by injection of antisense morpholino oligonucleotides has little or no effect on the embryo. In contrast, embryos that lack both vox and vent function have a dorsalized phenotype. Expression of dorsal mesodermal genes, including chordin, goosecoid and bozozok, is strongly expanded in embryos that lack vox and vent function, indicating that the redundant action of vox and vent is required to restrict dorsal genes to their appropriate territories. Our genetic analysis indicates that the dorsalizing transcription factor Bozozok promotes dorsal fates indirectly, by antagonizing the expression of vox and vent. In turn, vox and vent repress chordin expression, restricting its function as an antagonist of ventral fates to the dorsal side of the embryo. Our results support a model in which BMP signaling induces the expression of ventral genes, while vox and vent act redundantly to prevent the expression of chordin, goosecoid and other dorsal genes in the lateral and ventral mesendoderm. PMID:11493559

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

    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...

  4. Increasing the dynamic control space of mammalian transcription devices by combinatorial assembly of homologous regulatory elements from different bacterial species.

    Bacchus, William; Weber, Wilfried; Fussenegger, Martin

    2013-01-01

    Prokaryotic transcriptional regulatory elements are widely utilized building blocks for constructing regulatory genetic circuits adapted for mammalian cells and have found their way into a broad range of biotechnological applications. Prokaryotic transcriptional repressors, fused to eukaryotic transactivation or repression domains, compose the transcription factor, which binds and adjusts transcription from chimeric promoters containing the repressor-specific operator sequence. Escherichia coli and Chlamydia trachomatis share common features in the regulatory mechanism of the biosynthesis of l-tryptophan. The repressor protein TrpR of C. trachomatis regulates the trpRBA operon and the TrpR of E. coli regulates the trpEDCBA operon, both requiring l-tryptophan as a co-repressor. Fusion of these bacterial repressors to the VP16 transactivation domain of Herpes simplex virus creates synthetic transactivators that could bind and activate chimeric promoters, assembled by placing repressor-specific operator modules adjacent to a minimal promoter, in an l-tryptophan-adjustable manner. Combinations of different transactivator and promoter variants from the same or different bacterial species resulted in a multitude of regulatory systems where l-tryptophan regulation properties, background noise, and maximal gene expression levels were significantly diverse. Different l-tryptophan analogues showed diverse regulatory capacity depending on the promoter/transactivator combination. We believe the systems approach to rationally choose promoters, transactivators and inducer molecules, to obtain desired and predefined genetic expression dynamics and control profiles, will significantly advance the design of new regulatory circuits as well as improving already existing ones. PMID:23178502

  5. Selective modulation of promoter recruitment and transcriptional activity of PPARγ

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor regulated by the insulin-sensitizing thiazolidinediones (TZDs). We studied selective modulation of endogenous genes by PPARγ ligands using microarray, RNA expression kinetics, and chromatin immunoprecipitation (ChIP) in 3T3-L1 adipocytes. We found over 300 genes that were significantly regulated the TZDs pioglitazone, rosiglitazone, and troglitazone. TZD-mediated expression profiles were unique but overlapping. Ninety-one genes were commonly regulated by all three ligands. TZD time course and dose-response studies revealed gene- and TZD-specific expression kinetics. PEPCK expression was induced rapidly but PDK4 expression was induced gradually. Troglitazone EC50 values for PEPCK, PDK4, and RGS2 regulation were greater than those for pioglitazone and rosiglitazone. TZDs differentially induced histone acetylation of and PPARγ recruitment to target gene promoters. Selective modulation of PPARγ by TZDs resulted in distinct expression profiles and transcription kinetics which may be due to differential promoter activation and chromatin remodeling of target genes

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

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

    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

  8. The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

    Jenal, Mathias; Trinh, Emmanuelle; Britschgi, Christian;

    2009-01-01

    The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a...... the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell...

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

    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.

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

    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