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Sample records for cbf2 transcriptional activator

  1. JcCBF2 gene from Jatropha curcas improves freezing tolerance of Arabidopsis thaliana during the early stage of stress.

    Science.gov (United States)

    Wang, Linghui; Gao, Jihai; Qin, Xiaobo; Shi, Xiaodong; Luo, Lin; Zhang, Guozhen; Yu, Hongwu; Li, Chenyang; Hu, Minchao; Liu, Qifan; Xu, Ying; Chen, Fang

    2015-05-01

    High chilling-susceptibility is becoming the bottleneck for cultivation and commercialization of Jatropha curcas L. For insights to chilling resistance ability of this plant species, a cold response transcription factor, JcCBF2, was cloned and studied. It codes a 26 kDa protein, which contains all conserved motifs unique to the C-repeat binding factor (CBF) family and has high similarity to CBFs of Ricinus communis and Populus. Its transcripts express specifically in leaves of Jatropha at cold temperature. After transmitting the report vector, 35S::JcCBF2-GFP, into Arabidopsis thaliana, JcCBF2 protein is main detected in cell nucleus, being consistent to the nuclear orientation signal in its N-terminal. Compared to the control Arabidopsis, the frozen leaves of JcCBF2-overexpressed seedlings grow stronger with less malondialdehyde, smaller leaf conductivity and activer superoxide dismutase, showing their higher freezing tolerance. RT-PCR tests revealed that JcCBF2 functioned mainly at the early stage (0-6 h) of resistance events in Arabidopsis, and its transcripts reduced after 6 h. In addition, JcCBF2 could quickly regulate transcripts of some cold-responsive (COR) genes such as RD29A, COR105A and COR6.6, also during the early stage of frozen treatment. This study not only proves the chilling resistance roles of JcCBF2, but also presents a candidate gene engineering for improvement of chilling tolerance in J. curcas.

  2. Copy number variation at the HvCBF4-HvCBF2 genomic segment is a major component of frost resistance in barley.

    Science.gov (United States)

    Francia, Enrico; Morcia, Caterina; Pasquariello, Marianna; Mazzamurro, Valentina; Milc, Justyna Anna; Rizza, Fulvia; Terzi, Valeria; Pecchioni, Nicola

    2016-09-01

    A family of CBF transcription factors plays a major role in reconfiguring the plant transcriptome in response to low-freezing temperature in temperate cereals. In barley, more than 13 HvCBF genes map coincident with the major QTL FR-H2 suggesting them as candidates to explain the function of the locus. Variation in copy number (CNV) of specific HvCBFs was assayed in a panel of 41 barley genotypes using RT-qPCR. Taking advantage of an accurate phenotyping that combined Fv/Fm and field survival, resistance-associated variants within FR-H2 were identified. Genotypes with an increased copy number of HvCBF4 and HvCBF2 (at least ten and eight copies, respectively) showed greater frost resistance. A CAPS marker able to distinguish the CBF2A, CBF2B and CBF2A/B forms was developed and showed that all the higher-ranking genotypes in term of resistance harbour only CBF2A, while other resistant winter genotypes harbour also CBF2B, although at a lower CNV. In addition to the major involvement of the HvCBF4-HvCBF2 genomic segment in the proximal cluster of CBF elements, a negative role of HvCBF3 in the distal cluster was identified. Multiple linear regression models taking into account allelic variation at FR-H1/VRN-H1 explained 0.434 and 0.550 (both at p < 0.001) of the phenotypic variation for Fv/Fm and field survival respectively, while no interaction effect between CNV at the HvCBFs and FR-H1/VRN-H1 was found. Altogether our data suggest a major involvement of the CBF genes located in the proximal cluster, with no apparent involvement of the central cluster contrary to what was reported for wheat.

  3. Comparison of CBF1, CBF2, CBF3 and CBF4 expression in some grapevine cultivars and species under cold stress

    Science.gov (United States)

    Karimi, Maryam; Ebadi, Ali; Mousavi, Seyed Amir; Salami, Seyed Alireza; Zarei, Abdolkarim

    2015-01-01

    Grapevine, an important horticultural crop in the world, is moderately tolerant to cold conditions and is subjected to the cold injuries at different regions. So studies on different aspects of tolerance mechanism to unexpected cold of late spring as well as winter freezing seems necessary about this vine. For this reason, study on genes responsible for acquiring cold tolerance is very important. Transcription factors are among regulatory proteins that are responsible for cold acclimation. In this research work, expression levels of CBF1, CBF2, CBF3, and CBF4 transcription factors were studied on two cvs of Vitis vinifera (“Khalili-Danedar” and “Shahroodi”) as well as one Vitis riparia at different times after treating at 4 °C. Results showed that two vinifera cultivars, “Khalili-Danedar” and “Shahroodi”, had similar trend for each transcription factor. Gene expression increased at the beginning of cold stress and then decreased. Expression of these TF started some minutes (CBF1) after cold treatment and continued for several hours (CBF2), even till the tenth day (CBF4). All together V. riparia which is endemic to the cold regions behaved stronger and showed higher expression for all studied transcription factors. Among two V. vinifera cultivars, “Khalili-Danedar” showed significantly higher expression compared with “Shahroodi”. The comparison of expression levels of these four transcription factors revealed that the least and the greatest expressions were recorded for CBF1 and CBF3 respectively, and two CBF2 and CBF4 had approximately the same expression levels. PMID:26973374

  4. Analysis and Identification of Two CBF2 Gene Mutants in Arabidopsis%两种拟南芥 CBF2基因突变体的鉴定与分析

    Institute of Scientific and Technical Information of China (English)

    刘晓东; 刘超; 焦彬彬; 代培红; 苏秀娟; 李月

    2016-01-01

    Cold stress is one of the major abiotic stress,which limits the geographical distribution and yield of crops.CBF regulon plays a major role in freezing tolerance.CBF2,one of the components of the CBF regulon,has a negative role in freezing tolerance.In this study,two SALK mutants whose T-DNA insertion are both within the pro-moter of CBF2 gene,cbf2-1 and cbf2-2 were identified by qRT-PCR method.Results showed that the level of CBF2 gene expression in the two mutants reduced.CBF2 gene expression was induced by low temperature in cbf2-1 mut-nat.However,the degree of induction was lower in cbf2-1compared with the control.The effect of induction by low temperature was lost in cbf2-2.Analysis of freezing tolerance showed that the less degree of induction,the stronger freezing tolerance of plants and the higher the expression of downstream cold-responsive genes.More important thing was that although freezing resistance of the two CBF2 gene mutants were improved,their flowering time and seed yield per plant did not change significantly compared with the wild type plants.All the results indicate that CBF2 may be an ideal candidate target gene for crop breeding of freezing tolerance by genome editing technology.%低温是主要的逆境胁迫之一,限制了作物的地理分布和产量。筛选理想的抗冻靶基因用于分子育种对于稳定农业生产具有重要意义。CBF 调节子在植物抗冻反应中扮演主要的角色,CBF2是 CBF 调节子的组成成分之一,在抗冻反应中扮演负调控的作用。采用 qRT-PCR 和常规农艺性状测定法对2种 CBF2基因突变体 cbf2-1和 cbf2-2进行了多项指标的鉴定,结果发现在这2种突变体中 CBF2基因的表达存在不同程度的缺陷,cbf2-1中 CBF2基因受低温诱导表达,但表达量与对照相比,明显下降;而 cbf2-2中 CBF2基因诱导表达的效应完全丧失。抗冻性检测结果显示 CBF2基因的表达量越低,其突变体植株的抗冻性越强

  5. 抗冻基因CBF2表达载体构建及转化紫花苜蓿的研究%Construction of antifreeze gene CBF2 expression vector and transformation into alfalfa callus

    Institute of Scientific and Technical Information of China (English)

    刘晓静; 郝凤; 张德罡; 毛娟; 于铁峰

    2011-01-01

    Arabidopsis thaliana genomic DNA was selected as a template to amplify the target gene by PCR and connect it to the PGEM-T Easy Vector for construction of the T-CBF2. The target fragment and linear plasmids were obtained from the cloning vector T-CBF2 and from the plant expression vector PBI121 with dual digestion using BamH I and Sac I, respectively. The plant expression vector P-T-CBF2 was built through directional connections using T4 DNA ligase. PCR identification proved that the recombinant had been transferred into Agrobacterium tumefaciens and it was then introduced into Medicago sativa cv. Hetian through Agrobacterium-mediated transformation. Transfer of the target gene to alfalfa callus was successful.%本研究以拟南芥基因组DNA为模板,用PCR方法扩增目的基因,连接到PGEM-T Easy Vector载体上构建成克隆载体T-CBF2.用BamHI和Sacl分别对克隆载体T-CBF2和植物表达载体PBIl21进行双酶切,获得目的片段和线性质粒.在T4 DNA连接酶的作用下进行定向连接,构建成植物表达载体P-T-CBF2.采用直接转化法将重组子导入根癌农杆菌EHA105.经PCR鉴定,重组质粒已成功导入根癌农杆菌中.通过农杆菌介导法转化和田苜蓿,现在已经得到转基因的苜蓿愈伤组织.

  6. Sry is a transcriptional activator.

    Science.gov (United States)

    Dubin, R A; Ostrer, H

    1994-09-01

    The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

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

    Science.gov (United States)

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

    2015-11-05

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

  8. Isolation and Expression Analysis of Two Cold-Inducible Genes Encoding Putative CBF Transcription Factors from Chinese Cabbage (Brassica pekinensis Rupr.)

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Two homologous genes of the Arabidopsis C-repeat/dehydration-responsive element binding factors (CBF/DREB1) transcriptional activator were isolated by RT-PCR from Chinese cabbage (Brassica pekinensis Rupr.cv. Qinbai 5) and were designated as BcCBF1 and BcCBF2. Each encodes a putative CBF/DREB1 protein with an AP2 (Apetal2) DNA-binding domain, a putative nuclear localization signal, and a possible acidic activation domain. Deduced amino acid sequences show that BcCBF1 is very similar to the Arabidopsis CBF1, whereas BcCBF2 is different in that it contains two extra regions of 24 and 20 amino acids in the acidic domain. The mRNA accumulation profiles indicated that the expression of BcCBF1 and BcCBF2 is strongly induced by cold treatment, but does not respond similarly to dehydration or abscisic acid (ABA) treatment. However,the cold-induced accumulation of BcCBF2 mRNA was rapid but short-lived compared with that of BcCBF1.The mRNA levels of both BcCBF1 and BcCBF2 were higher in leaves than in roots when plants were exposed to cold, whereas, salt stress caused higher accumulation of BcCBF2 mRNA in roots than in leaves,suggesting that the organ specificity of the gene expression of the BcCBFs is probably stress dependent.In addition, the accumulation of BcCBF1 and BcCBF2 mRNAs was greatly enhanced by light compared with darkness when seedlings were exposed to cold. It is concluded that the two BcCBF proteins may be involved in the process of plant response to cold stress through an ABA-independent pathway and that there is also a cross-talk between the light signaling conduction pathway and the cold response pathway in B. pekinensis as In Arabidopsis.

  9. Rad51 activates polyomavirus JC early transcription.

    Directory of Open Access Journals (Sweden)

    Martyn K White

    Full Text Available The human neurotropic polyomavirus JC (JCV causes the fatal CNS demyelinating disease progressive multifocal leukoencephalopathy (PML. JCV infection is very common and after primary infection, the virus is able to persist in an asymptomatic state. Rarely, and usually only under conditions of immune impairment, JCV re-emerges to actively replicate in the astrocytes and oligodendrocytes of the brain causing PML. The regulatory events involved in the reactivation of active viral replication in PML are not well understood but previous studies have implicated the transcription factor NF-κB acting at a well-characterized site in the JCV noncoding control region (NCCR. NF-κB in turn is regulated in a number of ways including activation by cytokines such as TNF-α, interactions with other transcription factors and epigenetic events involving protein acetylation--all of which can regulate the transcriptional activity of JCV. Active JCV infection is marked by the occurrence of rapid and extensive DNA damage in the host cell and the induction of the expression of cellular proteins involved in DNA repair including Rad51, a major component of the homologous recombination-directed double-strand break DNA repair machinery. Here we show that increased Rad51 expression activates the JCV early promoter. This activation is co-operative with the stimulation caused by NF-κB p65, abrogated by mutation of the NF-κB binding site or siRNA to NFκB p65 and enhanced by the histone deacetylase inhibitor sodium butyrate. These data indicate that the induction of Rad51 resulting from infection with JCV acts through NF-κB via its binding site to stimulate JCV early transcription. We suggest that this provides a novel positive feedback mechanism to enhance viral gene expression during the early stage of JCV infection.

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Human cytomegalovirus IE2 protein interacts with transcription activating factors

    Institute of Scientific and Technical Information of China (English)

    XU; Jinping(徐进平); YE; Linbai(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.

  12. 巨桉EgrCBF1和EgrCBF2基因的克隆和胁迫响应表达分析%Cloning of EgrCBF1 and EgrCBF2 Genes from Eucalyptus grandis and their Expression under Stresses

    Institute of Scientific and Technical Information of China (English)

    王京京; 童再康; 黄程前; 程龙军

    2012-01-01

    Two CBF genes named EgrCBFl and EgrCBF2 ( GenBank No. JQ068827 ; JQ068828 ) were isolated from Eucalyptus grandis seedlings under low temperature. Sequence analysis showed that EgrCBFl and EgrCBFl cDNA had 1 062 bp and 1 203 bp, respectively. The two genes encoded 220 and 196 amino acids, separately and both contained an AP2 domain. The results of homology comparison showed that EgrCBFl and EgrCBF2 protein shared 91% and 80% homology with EguCBFla. Spatial expression analyses showed that EgrCBFl expressed mainly in leaves and roots while EgrCBF2 expressed in all leaves, stems and roots. qRT-PCR result of EgrCBFl and EgrCBFl under 0 ℃ ,2 ℃ ,4 ℃ ,6 ℃ , and 8 ℃ revealed that they were both induced by low temperatures and reached the highest level at 2 ℃ . Time course of the genes expressions at 4 ℃ for 2 h,4 h,8 h,24 h,and 48 h showed that their expressions increased at the first and then decreased. Changes in EgrCBFl and EgrCBFl expression under 100 μmol · L-1 ABA, 200 mmol·L-1 NaCl and drought were also analyzed. The results showed EgrCBFl was up-regulated under drought stress and ABA treatment, while the expression of EgrCBF2 increased under drought and salt stress.%从低温诱导的巨桉幼苗中克隆到2条CBF的全长cDNA序列,命名为EgrCBF1和EgrCBF2,全长分别为1 062 bp和1 203 bp,编码220个氨基酸和196个氨基酸,命名为EgrCBF1和EgrCBF2(GenBank登录号分别为:JQ068827; JQ068828),都包含1个AP2结构域.2个基因编码的蛋白都与冈尼桉中CBF蛋白(DQ241820)具有很高的同源性,与EguCBF1a的同源性分别达到了91%和80%.RT-PCR分析表明,EgrCBF1主要在叶和根中表达,而EgrCBF2在叶、茎和根中都有表达.对不同低温条件(0,2,4,6,8℃)和4℃2,4,8,24,48 h处理下EgrCBF1和EgrCBF2的qRT-PCR分析表明:2个基因都受低温诱导,并在2℃时诱导水平达到最高;4℃下随低温时间的延长,它们的诱导表达特性都呈先升后降的趋势.在100 μmol·L-1 ABA,200 mmol

  13. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

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

    2012-01-01

    on 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...... base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short...

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

    Science.gov (United States)

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

    2010-10-01

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

  15. A new type of NtrC transcriptional activator.

    OpenAIRE

    Foster-Hartnett, D; Cullen, P. J.; Monika, E M; Kranz, R G

    1994-01-01

    The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the sigma 54 factor. Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (> 100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for a...

  16. Transcriptional activators enhance polyadenylation of mRNA precursors

    OpenAIRE

    Nagaike, Takashi; Manley, James L.

    2011-01-01

    3′ processing of mRNA precursors is frequently coupled to transcription by RNA polymerase II (RNAP II). This coupling is well known to involve the C-terminal domain of the RNAP II largest subunit, but a variety of other transcription-associated factors have also been suggested to mediate coupling. Our recent studies have provided direct evidence that transcriptional activators can enhance the efficiency of transcription-coupled 3′ processing. In this point-of-view, we discuss the mechanisms t...

  17. Human mediator subunit MED15 promotes transcriptional activation.

    Science.gov (United States)

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

    2014-10-01

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

  18. Transcriptional Activation of the Zygotic Genome in Drosophila.

    Science.gov (United States)

    Harrison, Melissa M; Eisen, Michael B

    2015-01-01

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

  19. Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

    Science.gov (United States)

    Hamard, Pierre-Jacques; Boyer-Guittaut, Michaël; Camuzeaux, Barbara; Dujardin, Denis; Hauss, Charlotte; Oelgeschläger, Thomas; Vigneron, Marc; Kedinger, Claude; Chatton, Bruno

    2007-01-01

    Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

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

    Science.gov (United States)

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

    2012-06-01

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

  1. Dataset of transcriptional landscape of B cell early activation

    Directory of Open Access Journals (Sweden)

    Alexander S. Garruss

    2015-09-01

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

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

    Science.gov (United States)

    Kobrinsky, Evgeny

    2015-01-01

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

  3. Potential Role of Activating Transcription Factor 5 during Osteogenesis

    Directory of Open Access Journals (Sweden)

    Luisa Vicari

    2016-01-01

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

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

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

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

  5. First Exon Length Controls Active Chromatin Signatures and Transcription

    Directory of Open Access Journals (Sweden)

    Nicole I. Bieberstein

    2012-07-01

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

  6. Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors.

    Science.gov (United States)

    Thiel, Gerald; Rössler, Oliver G

    2017-03-01

    Resveratrol (trans-3,4',5-trihydroxystilbene), a polyphenolic phytoalexin of grapes and other fruits and plants, is a common constituent of our diet and of dietary supplements. Many health-promoting benefits have been connected with resveratrol in the treatment of cardiovascular diseases, cancer, diabetes, inflammation, neurodegeneration, and diseases connected with aging. To explain the pleiotropic effects of resveratrol, the molecular targets of this compound have to be identified on the cellular level. Resveratrol induces intracellular signal transduction pathways which ultimately lead to changes in the gene expression pattern of the cells. Here, we review the effect of resveratrol on the activation of the stimulus-responsive transcription factors CREB, AP-1, Egr-1, Elk-1, and Nrf2. Following activation, these transcription factors induce transcription of delayed response genes. The gene products of these delayed response genes are ultimately responsible for the changes in the biochemistry and physiology of resveratrol-treated cells. The activation of stimulus-responsive transcription factors may explain many of the intracellular activities of resveratrol. However, results obtained in vitro may not easily be transferred to in vivo systems.

  7. Transcriptional activity of transposable elements in coelacanth.

    Science.gov (United States)

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

    2014-09-01

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

  8. Genome-wide signatures of transcription factor activity: connecting transcription factors, disease, and small molecules.

    Directory of Open Access Journals (Sweden)

    Jing Chen

    Full Text Available Identifying transcription factors (TF involved in producing a genome-wide transcriptional profile is an essential step in building mechanistic model that can explain observed gene expression data. We developed a statistical framework for constructing genome-wide signatures of TF activity, and for using such signatures in the analysis of gene expression data produced by complex transcriptional regulatory programs. Our framework integrates ChIP-seq data and appropriately matched gene expression profiles to identify True REGulatory (TREG TF-gene interactions. It provides genome-wide quantification of the likelihood of regulatory TF-gene interaction that can be used to either identify regulated genes, or as genome-wide signature of TF activity. To effectively use ChIP-seq data, we introduce a novel statistical model that integrates information from all binding "peaks" within 2 Mb window around a gene's transcription start site (TSS, and provides gene-level binding scores and probabilities of regulatory interaction. In the second step we integrate these binding scores and regulatory probabilities with gene expression data to assess the likelihood of True REGulatory (TREG TF-gene interactions. We demonstrate the advantages of TREG framework in identifying genes regulated by two TFs with widely different distribution of functional binding events (ERα and E2f1. We also show that TREG signatures of TF activity vastly improve our ability to detect involvement of ERα in producing complex diseases-related transcriptional profiles. Through a large study of disease-related transcriptional signatures and transcriptional signatures of drug activity, we demonstrate that increase in statistical power associated with the use of TREG signatures makes the crucial difference in identifying key targets for treatment, and drugs to use for treatment. All methods are implemented in an open-source R package treg. The package also contains all data used in the analysis

  9. Exploring cellular memory molecules marking competent and active transcriptions

    Directory of Open Access Journals (Sweden)

    Liu De-Pei

    2007-05-01

    Full Text Available Abstract Background Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation. Results We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription. Conclusion Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

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

    Science.gov (United States)

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

    2015-03-18

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

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

    Science.gov (United States)

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

    2015-06-18

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

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

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  14. [Transcription activator-like effectors(TALEs)based genome engineering].

    Science.gov (United States)

    Zhao, Mei-Wei; Duan, Cheng-Li; Liu, Jiang

    2013-10-01

    Systematic reverse-engineering of functional genome architecture requires precise modifications of gene sequences and transcription levels. The development and application of transcription activator-like effectors(TALEs) has created a wealth of genome engineering possibilities. TALEs are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas species. The DNA-binding domain of each TALE typically consists of tandem 34-amino acid repeat modules rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Such "genome engineering" has now been established in human cells and a number of model organisms, thus opening the door to better understanding gene function in model organisms, improving traits in crop plants and treating human genetic disorders.

  15. Human DJ-1-specific Transcriptional Activation of Tyrosine Hydroxylase Gene*

    Science.gov (United States)

    Ishikawa, Shizuma; Taira, Takahiro; Takahashi-Niki, Kazuko; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M. M.

    2010-01-01

    Loss-of-function mutation in the DJ-1 gene causes a subset of familial Parkinson disease. The mechanism underlying DJ-1-related selective vulnerability in the dopaminergic pathway is, however, not known. DJ-1 has multiple functions, including transcriptional regulation, and one of transcriptional target genes for DJ-1 is the tyrosine hydroxylase (TH) gene, the product of which is a key enzyme for dopamine biosynthesis. It has been reported that DJ-1 is a neuroprotective transcriptional co-activator that sequesters a transcriptional co-repressor polypyrimidine tract-binding protein-associated splicing factor (PSF) from the TH gene promoter. In this study, we found that knockdown of human DJ-1 by small interference RNA in human dopaminergic cell lines attenuated TH gene expression and 4-dihydroxy-l-phenylalanine production but that knockdown or knock-out of mouse DJ-1 in mouse cell lines or in mice did not affect such expression and TH activity. In reporter assays using the human TH gene promoter linked to the luciferase gene, stimulation of TH promoter activity was observed in human cells, but not mouse cells, that had been transfected with DJ-1. Although human DJ-1 and mouse DJ-1 were associated either with human or with mouse PSF, TH promoter activity inhibited by PSF was restored by human DJ-1 but not by mouse DJ-1. Chromatin immunoprecipitation assays revealed that the complex of PSF with DJ-1 bound to the human but not the mouse TH gene promoter. These results suggest a novel species-specific transcriptional regulation of the TH promoter by DJ-1 and one of the mechanisms for no reduction of TH in DJ-1-knock-out mice. PMID:20938049

  16. A transcription activator-like effector toolbox for genome engineering.

    Science.gov (United States)

    Sanjana, Neville E; Cong, Le; Zhou, Yang; Cunniff, Margaret M; Feng, Guoping; Zhang, Feng

    2012-01-05

    Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas sp. The DNA-binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox described here will enable a broad range of biological applications.

  17. Activating transcription factor 4 regulates osteoclast differentiation in mice.

    Science.gov (United States)

    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; Xiao, Guozhi

    2010-08-01

    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 to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity.

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

    Science.gov (United States)

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

    2011-04-01

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

  19. Activating transcription factor 4 regulates osteoclast differentiation in mice

    Science.gov (United States)

    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; Xiao, Guozhi

    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 to restore the OCL differentiation defect. Conversely, Trap-Atf4-tg mice displayed severe osteopenia with dramatically increased osteoclastogenesis and bone resorption. We further showed that ATF4 was an upstream activator of the critical transcription factor Nfatc1 and was critical for RANKL activation of multiple MAPK pathways in OCL progenitors. Furthermore, ATF4 was crucial for M-CSF induction of RANK expression on BMMs, and lack of ATF4 caused a shift in OCL precursors to macrophages. Finally, ATF4 was largely modulated by M-CSF signaling and the PI3K/AKT pathways in BMMs. These results demonstrate that ATF4 plays a direct role in regulating OCL differentiation and suggest that it may be a therapeutic target for treating bone diseases associated with increased OCL activity. PMID:20628199

  20. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

    DEFF Research Database (Denmark)

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

    2016-01-01

    Whole-cell biocatalysts have proven a tractable path toward sustainable production of bulk and fine chemicals. Yet the screening of libraries of cellular designs to identify best-performing biocatalysts is most often a low-throughput endeavor. For this reason, the development of biosensors enabling...... real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily...... of LysR-type transcriptional regulators (LTTRs). We identified a design supporting LTTR-dependent activation of reporter gene expression in the presence of cognate small-molecule inducers. As proof of principle, we applied the biosensors for in vivo screening of cells producing naringenin or cis...

  1. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

    Directory of Open Access Journals (Sweden)

    Dinesh K. Singh

    2017-01-01

    Full Text Available Efforts to identify and target glioblastoma (GBM drivers have primarily focused on receptor tyrosine kinases (RTKs. Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2 transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2 and zinc-finger E-box binding homeobox 1 (ZEB1, which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

  2. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma.

    Science.gov (United States)

    Singh, Dinesh K; Kollipara, Rahul K; Vemireddy, Vamsidara; Yang, Xiao-Li; Sun, Yuxiao; Regmi, Nanda; Klingler, Stefan; Hatanpaa, Kimmo J; Raisanen, Jack; Cho, Steve K; Sirasanagandla, Shyam; Nannepaga, Suraj; Piccirillo, Sara; Mashimo, Tomoyuki; Wang, Shan; Humphries, Caroline G; Mickey, Bruce; Maher, Elizabeth A; Zheng, Hongwu; Kim, Ryung S; Kittler, Ralf; Bachoo, Robert M

    2017-01-24

    Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2) transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc-finger E-box binding homeobox 1 (ZEB1), which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

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

    Science.gov (United States)

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

    2009-09-16

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

  4. Identification of Post-Transcriptional Modulators of Breast Cancer Transcription Factor Activity Using MINDy

    Science.gov (United States)

    Campbell, Thomas M.; Castro, Mauro A. A.; Ponder, Bruce A. J.

    2016-01-01

    We have recently identified transcription factors (TFs) that are key drivers of breast cancer risk. To better understand the pathways or sub-networks in which these TFs mediate their function we sought to identify upstream modulators of their activity. We applied the MINDy (Modulator Inference by Network Dynamics) algorithm to four TFs (ESR1, FOXA1, GATA3 and SPDEF) that are key drivers of estrogen receptor-positive (ER+) breast cancer risk, as well as cancer progression. Our computational analysis identified over 500 potential modulators. We assayed 189 of these and identified 55 genes with functional characteristics that were consistent with a role as TF modulators. In the future, the identified modulators may be tested as potential therapeutic targets, able to alter the activity of TFs that are critical in the development of breast cancer. PMID:27997592

  5. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

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

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

    Science.gov (United States)

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

    2010-01-01

    Vascular endothelial growth factor (VEGF) is required for proper lung development and is transcriptionally regulated in alveolar epithelial cells by hypoxia inducible factor (HIF). Previous findings in a newborn mouse model of bronchopulmonary dysplasia (BPD) suggest that thioredoxin interacting protein (Txnip) is a novel regulator of VEGF expression. The present studies were designed to test the hypothesis that Txnip negatively regulates VEGF through effects on HIF-mediated gene expression. To test this hypothesis, we first examined the levels of VEGF and Txnip protein in the lungs of 1 day-old newborn and E19 embryos and detected a significant inverse correlation. To elucidate the mechanisms underlying this relationship, we studied the effects of Txnip overexpression on HIF-mediated transcription using murine lung epithelial (MLE-12) cells. Overexpression of Txnip inhibited HIF-mediated reporter activity in both hypoxia and room air. Suppression of HIF activity by Txnip appeared to be independent of the ability of Txnip to bind to thioredoxin. Thus, our studies support a model in which Txnip is a potentially critical regulator of HIF-mediated gene transcription in the murine lung. Alterations in Txnip expression could alter lung VEGF expression in prematurely born human infants and contribute to the development of BPD. PMID:20692333

  7. An Ikaros Promoter Element with Dual Epigenetic and Transcriptional Activities.

    Science.gov (United States)

    Perotti, Elizabeth A; Georgopoulos, Katia; Yoshida, Toshimi

    2015-01-01

    Ikaros DNA binding factor plays critical roles in lymphocyte development. Changes in Ikaros expression levels during lymphopoiesis are controlled by redundant but also unique regulatory elements of its locus that are critical for this developmental process. We have recently shown that Ikaros binds its own locus in thymocytes in vivo. Here, we evaluated the role of an Ikaros binding site within its major lympho-myeloid promoter. We identified an Ikaros/Ets binding site within a promoter sub-region that was highly conserved in mouse and human. Deletion of this binding site increased the percentage of the reporter-expressing mouse lines, indicating that its loss provided a more permissive chromatin environment. However, once transcription was established, the lack of this site decreased transcriptional activity. These findings implicate a dual role for Ikaros/Ets1 binding on Ikzf1 expression that is exerted at least through its promoter.

  8. An Ikaros Promoter Element with Dual Epigenetic and Transcriptional Activities.

    Directory of Open Access Journals (Sweden)

    Elizabeth A Perotti

    Full Text Available Ikaros DNA binding factor plays critical roles in lymphocyte development. Changes in Ikaros expression levels during lymphopoiesis are controlled by redundant but also unique regulatory elements of its locus that are critical for this developmental process. We have recently shown that Ikaros binds its own locus in thymocytes in vivo. Here, we evaluated the role of an Ikaros binding site within its major lympho-myeloid promoter. We identified an Ikaros/Ets binding site within a promoter sub-region that was highly conserved in mouse and human. Deletion of this binding site increased the percentage of the reporter-expressing mouse lines, indicating that its loss provided a more permissive chromatin environment. However, once transcription was established, the lack of this site decreased transcriptional activity. These findings implicate a dual role for Ikaros/Ets1 binding on Ikzf1 expression that is exerted at least through its promoter.

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

    Bhaumik, Sukesh R

    2011-02-01

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

  12. Rethinking transcriptional activation in the Arabidopsis circadian clock.

    Directory of Open Access Journals (Sweden)

    Karl Fogelmark

    2014-07-01

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

  13. Rethinking transcriptional activation in the Arabidopsis circadian clock.

    Science.gov (United States)

    Fogelmark, Karl; Troein, Carl

    2014-07-01

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

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

    Science.gov (United States)

    Chang, Ji Suk; Gettys, Thomas W

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

  16. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Ludmila R.P. Ferreira

    2008-03-01

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

  17. Activator protein 1 promotes the transcriptional activation of IRAK-M.

    Science.gov (United States)

    Jin, Peipei; Bo, Lulong; Liu, Yongjian; Lu, Wenbin; Lin, Shengwei; Bian, Jinjun; Deng, Xiaoming

    2016-10-01

    Interleukin-1 receptor-associated kinase M (IRAK-M) is a well-known negative regulator for Toll-like receptor signaling, which can regulate immune homeostasis and tolerance in a number of pathological settings. However, the mechanism for IRAK-M regulation at transcriptional level remains largely unknown. In this study, a 1.4kb upstream sequence starting from the major IRAK-M transcriptional start site was cloned into luciferase reporter vector pGL3-basic to construct the full-length IRAK-M promoter. Luciferase reporter plasmids harboring the full-length and the deletion mutants of IRAK-M were transfected into 293T and A549 cells, and their relative luciferase activity was measured. The results demonstrated that activator protein 1(AP-1) cis-element plays a crucial role in IRAK-M constitutive gene transcription. Silencing of c-Fos and/or c-Jun expression suppressed the IRAK-M promoter activity as well as its mRNA and protein expressions. As a specific inhibitor for AP-1 activation, SP600125 also significantly suppressed the basal transcriptional activity of IRAK-M, the binding activity of c-Fos/c-Jun with IRAK-M promoter, and IRAK-M protein expression. Taken together, the result of this study highlights the importance of AP-1 in IRAK-M transcription, which offers more information on the role of IRAK-M in infectious and non-infectious diseases.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-17

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

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

    Directory of Open Access Journals (Sweden)

    Bol John F

    2011-05-01

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

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

    DEFF Research Database (Denmark)

    Mourier, Tobias; Willerslev, Eske

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Juan Zhang

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    DEFF Research Database (Denmark)

    Mourier, Tobias; Willerslev, Eske

    2010-01-01

    of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes. CONCLUSIONS: Presumably, the host organism negatively regulates...

  5. SUMO modification regulates the transcriptional activity of FLASH

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-09-25

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

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

    Science.gov (United States)

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

    2009-01-23

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

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

    Science.gov (United States)

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

    2015-11-11

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Lai Meng-Jiun

    2010-08-01

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

  11. Transcription factor PIF4 controls the thermosensory activation of flowering

    KAUST Repository

    Kumar, S. Vinod

    2012-03-21

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

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

    Science.gov (United States)

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

    2012-10-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2014-03-01

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

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

    NARCIS (Netherlands)

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

    2014-01-01

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

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

    Science.gov (United States)

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

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

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

    Science.gov (United States)

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

  18. Identification,characterization and expression analysis of transcription factor (CBF) genes in rice (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    Yunfei CAO; Jiaojiao WANG; Li GUO; Kai XIAO

    2008-01-01

    The acclimation of plants to cold,salt and dehydration is involved in the action of the transcription factor (CBF) cold-response pathway.In this paper,nineteen rice CBF genes,including seven previously released and twelve unpublished novels,were identified and characterized.The multi-members of rice CBFs (OsCBF1 to OsCBF12) were divergent at the nucleotide and amino acid level.Expression analysis shows that five novel rice CBF genes (OsCBF1,OsCBF2,OsCBF3,OsCBF8,and OsCBF9) responded to short-term (1 h or 3 h) stresses of low temperature,salt stress and dehydration.The transcripts of OsCBF2,OsCBF8 and OsCBF9 in the roots were rapidly elevated when the plants were exposed to low temperatures,suggesting that they were possibly involved in low temperature responses in rice plants.Meanwhile,the expression level of OsCBF2 in leaves was enhanced when exposed to salt stress of 1-3 h,implying that OsCBF2 functioned as a transduction component in the salt stress signal cascade.Various expression patterns in OsCBF1,OsCBF2,OsCBF3,OsCBF8,and OsCBF9 under low temperature,salt and drought conditions,together with the different expression patterns between roots and leaves for each of these indicated that every rice CBF gene has unique and non-redundant functions in the response to the abiotic stresses.

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Science.gov (United States)

    Ha, Changhoon; Lim, Kihong

    2015-11-13

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

  1. Altered activities of transcription factors and their related gene expression in cardiac tissues of diabetic rats.

    Science.gov (United States)

    Nishio, Y; Kashiwagi, A; Taki, H; Shinozaki, K; Maeno, Y; Kojima, H; Maegawa, H; Haneda, M; Hidaka, H; Yasuda, H; Horiike, K; Kikkawa, R

    1998-08-01

    Gene regulation in the cardiovascular tissues of diabetic subjects has been reported to be altered. To examine abnormal activities in transcription factors as a possible cause of this altered gene regulation, we studied the activity of two redox-sensitive transcription factors--nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1)--and the change in the mRNA content of heme oxygenase-1, which is regulated by these transcription factors in the cardiac tissues of rats with streptozotocin-induced diabetes. Increased activity of NF-kappaB and AP-1 but not nuclear transcription-activating factor, as determined by an electrophoretic mobility shift assay, was found in the hearts of 4-week diabetic rats. Glycemic control by a subcutaneous injection of insulin prevented these diabetes-induced changes in transcription factor activity. In accordance with these changes, the mRNA content of heme oxygenase-1 was increased fourfold in 4-week diabetic rats and threefold in 24-week diabetic rats as compared with control rats (P oxidative stress is involved in the activation of the transcription factors NF-kappaB and AP-1 in the cardiac tissues of diabetic rats, and that these abnormal activities of transcription factors could be associated with the altered gene regulation observed in the cardiovascular tissues of diabetic rats.

  2. pilS loci in Neisseria gonorrhoeae are transcriptionally active

    Science.gov (United States)

    Wachter, Jenny; Masters, Thao L.; Wachter, Shaun; Mason, Joanna

    2015-01-01

    Piliation is an important virulence determinant for Neisseria gonorrhoeae. PilE polypeptide is the major protein subunit in the pilus organelle and engages in extensive antigenic variation due to recombination between pilE and a pilS locus. pilS were so-named as they are believed to be transcriptionally silent, in contrast to the pilE locus. In this study, we demonstrate the presence of a small, pil-specific RNA species. Through using a series of pilE deletion mutants, we show by Northern blotting and quantitative reverse transcriptase PCR analysis (qRT-PCR), that these smaller RNA species are not derived from the primary pilE transcript following some processing events, but rather, arose through transcription of the pilS loci. Small transcriptome analysis, in conjunction with analysis of pilS recombinants, identified both sense and anti-sense RNAs originating from most, but not all, of the pilS gene copies. Focusing on the MS11 pilS6 locus, we identified by site-directed mutagenesis a sense promoter located immediately upstream of pilS6 copy 2, as well as an anti-sense promoter immediately downstream of pilS6 copy 1. Whole transcriptome analysis also revealed the presence of pil-specific sRNA in both gonococci and meningococci. Overall, this study reveals an added layer of complexity to the pilE/pilS recombination scheme by demonstrating pil-specific transcription within genes that were previously thought to be transcriptionally silent. PMID:25701734

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

    Directory of Open Access Journals (Sweden)

    Galbraith Simon J

    2005-06-01

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

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

    Science.gov (United States)

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

    2010-12-14

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

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

    Science.gov (United States)

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

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

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

    Science.gov (United States)

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

    2014-01-24

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

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

    NARCIS (Netherlands)

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

    2003-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-04

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

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2014-04-01

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

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

    Science.gov (United States)

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

    2014-06-27

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

  13. Double-Stranded-RNA-Activated Protein Kinase PKR Enhances Transcriptional Activation by Tumor Suppressor p53

    OpenAIRE

    1999-01-01

    The tumor suppressor p53 plays a key role in inducing G1 arrest and apoptosis following DNA damage. The double-stranded-RNA-activated protein PKR is a serine/threonine interferon (IFN)-inducible kinase which plays an important role in regulation of gene expression at both transcriptional and translational levels. Since a cross talk between IFN-inducible proteins and p53 had already been established, we investigated whether and how p53 function was modulated by PKR. We analyzed p53 function in...

  14. Gene expression profiling of aging reveals activation of a p53-mediated transcriptional program

    Directory of Open Access Journals (Sweden)

    Weindruch Richard

    2007-03-01

    Full Text Available Abstract Background Aging has been associated with widespread changes at the gene expression level in multiple mammalian tissues. We have used high density oligonucleotide arrays and novel statistical methods to identify specific transcriptional classes that may uncover biological processes that play a central role in mammalian aging. Results We identified 712 transcripts that are differentially expressed in young (5 month old and old (25-month old mouse skeletal muscle. Caloric restriction (CR completely or partially reversed 87% of the changes in expression. Examination of individual genes revealed a transcriptional profile indicative of increased p53 activity in the older muscle. To determine whether the increase in p53 activity is associated with transcriptional activation of apoptotic targets, we performed RT-PCR on four well known mediators of p53-induced apoptosis: puma, noxa, tnfrsf10b and bok. Expression levels for these proapoptotic genes increased significantly with age (P +/- and GPX4+/- mice, suggesting that oxidative stress does not induce the expression of these genes. Western blot analysis confirmed that protein levels for both p21 and GADD45a, two established transcriptional targets of p53, were higher in the older muscle tissue. Conclusion These observations support a role for p53-mediated transcriptional program in mammalian aging and suggest that mechanisms other than reactive oxygen species are involved in the age-related transcriptional activation of p53 targets.

  15. Global identification and characterization of transcriptionally active regions in the rice genome.

    Directory of Open Access Journals (Sweden)

    Lei Li

    Full Text Available Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs not encoded by annotated exons in the rice (Oryza. sativa subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83% japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome.

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

    Science.gov (United States)

    Kim, Kihoon; Kim, AeRi

    2010-09-01

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

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

    Science.gov (United States)

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

    2011-10-01

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

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

    Science.gov (United States)

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

    2015-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Jakob Madsen Pedersen

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

  20. Ataxia telangiectasia mutated activation by transcription- and topoisomerase I-induced DNA double-strand breaks.

    Science.gov (United States)

    Sordet, Olivier; Redon, Christophe E; Guirouilh-Barbat, Josée; Smith, Susan; Solier, Stéphanie; Douarre, Céline; Conti, Chiara; Nakamura, Asako J; Das, Benu B; Nicolas, Estelle; Kohn, Kurt W; Bonner, William M; Pommier, Yves

    2009-08-01

    Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

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

    Science.gov (United States)

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

    2015-01-01

    Mitochondrial dysfunction has emerged as an important factor in wide ranging human pathologies. We have previously defined a retrograde signaling pathway that originates from dysfunctional mitochondria (Mt-RS) and causes a global nuclear transcriptional reprograming as its endpoint. Mitochondrial dysfunction causing disruption of mitochondrial membrane potential and consequent increase in cytosolic calcium [Ca2](c) activates calcineurin and the transcription factors NF-κB, NFAT, CREB, and C/EBPδ. In macrophages this signaling complements receptor activator of nuclear factor kappa-B ligand (RANKL)–induced osteoclastic differentiation. Here, we show that the Mt-RS activated transcriptional coactivator heterogeneous ribonucleoprotein A2 (hnRNP A2) is induced by hypoxia in murine macrophages. We demonstrate that the cathepsin K gene (Cstk), one of the key genes upregulated during osteoclast differentiation, is transcriptionally activated by Mt-RS factors. HnRNP A2 acts as a coactivator with nuclear transcription factors, cRel, and C/EBPδ for Cstk promoter activation under hypoxic conditions. Notably, our study shows that hypoxia-induced activation of the stress target factors mediates effects similar to that of RANKL with regard to Cstk activation. We therefore suggest that mitochondrial dysfunction and activation of Mt-RS, induced by various pathophysiologic conditions, is a potential risk factor for osteoclastogenesis and bone loss. PMID:25800988

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Huan Qiao

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

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

    Science.gov (United States)

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

    2015-10-01

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

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

    NARCIS (Netherlands)

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

    1992-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Julia K Bialek

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

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

    Science.gov (United States)

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

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the underlying molecular mechanisms. Agonist-activated FXR inhibits glucose-induced transcription of several glycolytic genes, including the liver-type pyruvate kinase gene (L-PK), in the immortalized human hepatocyte (IHH) and HepaRG cell lines. This inhibition requires the L4L3 region of the L-PK promoter, known to bind the transcription factors ChREBP and hepatocyte nuclear factor 4α (HNF4α). FXR interacts directly with ChREBP and HNF4α proteins. Analysis of the protein complex bound to the L4L3 region reveals the presence of ChREBP, HNF4α, FXR, and the transcriptional coactivators p300 and CBP at high glucose concentrations. FXR activation does not affect either FXR or HNF4α binding to the L4L3 region but does result in the concomitant release of ChREBP, p300, and CBP and in the recruitment of the transcriptional corepressor SMRT. Thus, FXR transrepresses the expression of genes involved in glycolysis in human hepatocytes. PMID:23530060

  8. DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions.

    Science.gov (United States)

    Sordet, Olivier; Nakamura, Asako J; Redon, Christophe E; Pommier, Yves

    2010-01-15

    A taxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA double-strand break (DSB) response. We recently showed that transcription-blocking topoisomerase I cleavage complexes (TOP1cc) produce DSBs related to R-loop formation and activate ATM in post-mitotic neurons and lymphocytes. Here we discuss how TOP1cc can produce transcription arrest with R-loop formation and generate DSBs that activate ATM, as well as data suggesting that those transcription-dependent DSBs tend to form at the IgH locus and at specific genomic sites. We also address the potential roles of ATM in response to transcription-blocking TOP1cc.

  9. Proximal genomic localization of STAT1 binding and regulated transcriptional activity

    Directory of Open Access Journals (Sweden)

    Smyth Gordon K

    2006-10-01

    Full Text Available Abstract Background Signal transducer and activator of transcription (STAT proteins are key regulators of gene expression in response to the interferon (IFN family of anti-viral and anti-microbial cytokines. We have examined the genomic relationship between STAT1 binding and regulated transcription using multiple tiling microarray and chromatin immunoprecipitation microarray (ChIP-chip experiments from public repositories. Results In response to IFN-γ, STAT1 bound proximally to regions of the genome that exhibit regulated transcriptional activity. This finding was consistent between different tiling microarray platforms, and between different measures of transcriptional activity, including differential binding of RNA polymerase II, and differential mRNA transcription. Re-analysis of tiling microarray data from a recent study of IFN-γ-induced STAT1 ChIP-chip and mRNA expression revealed that STAT1 binding is tightly associated with localized mRNA transcription in response to IFN-γ. Close relationships were also apparent between STAT1 binding, STAT2 binding, and mRNA transcription in response to IFN-α. Furthermore, we found that sites of STAT1 binding within the Encyclopedia of DNA Elements (ENCODE region are precisely correlated with sites of either enhanced or diminished binding by the RNA polymerase II complex. Conclusion Together, our results indicate that STAT1 binds proximally to regions of the genome that exhibit regulated transcriptional activity. This finding establishes a generalized basis for the positioning of STAT1 binding sites within the genome, and supports a role for STAT1 in the direct recruitment of the RNA polymerase II complex to the promoters of IFN-γ-responsive genes.

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

    Science.gov (United States)

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

    2011-10-11

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

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

    KAUST Repository

    Roy, S.

    2015-06-27

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

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

    Directory of Open Access Journals (Sweden)

    Jolanta Kwasniewska

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

  13. DnaA-stimulated transcriptional activation of oriλ: Escherichia coli RNA polymerase β subunit as a transcriptional activator contact site

    OpenAIRE

    Szalewska-Palasz, Agnieszka; Wegrzyn, Alicja; Blaszczak, Adam; Taylor, Karol; Wegrzyn, Grzegorz

    1998-01-01

    We present evidence that Escherichia coli RNA polymerase β subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage λ. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of λ plasmids; this suppression was allele-specific. When a pote...

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

    Science.gov (United States)

    Thakore, Pratiksha I; Gersbach, Charles A

    2016-01-01

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

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

    Science.gov (United States)

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

    2012-07-01

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

  16. Transcriptional Activity of Human Endogenous Retroviruses in Human Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Emanuela Balestrieri

    2015-01-01

    Full Text Available Human endogenous retroviruses (HERVs have been implicated in human physiology and in human pathology. A better knowledge of the retroviral transcriptional activity in the general population and during the life span would greatly help the debate on its pathologic potential. The transcriptional activity of four HERV families (H, K, W, and E was assessed, by qualitative and quantitative PCR, in PBMCs from 261 individuals aged from 1 to 80 years. Our results show that HERV-H, HERV-K, and HERV-W, but not HERV-E, are transcriptionally active in the test population already in the early childhood. In addition, the transcriptional levels of HERV-H, HERV-K, and HERV-W change significantly during the life span, albeit with distinct patterns. Our results, reinforce the hypothesis of a physiological correlation between HERVs activity and the different stages of life in humans. Studies aiming at identifying the factors, which are responsible for these changes during the individual’s life, are still needed. Although the observed phenomena are presumably subjected to great variability, the basal transcriptional activity of each individual, also depending on the different ages of life, must be carefully considered in all the studies involving HERVs as causative agents of disease.

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

    Directory of Open Access Journals (Sweden)

    Carmelo Ferrai

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tsai Chueh-Jen

    2010-01-01

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

  19. Differences in transcriptional activity of cutaneous human papillomaviruses

    DEFF Research Database (Denmark)

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

    2008-01-01

    The interaction between UV-B irradiation and cutaneous human papillomaviruses (HPV) has been suggested to be of relevance for the development of non-melanoma skin cancers. We investigated the activity within the upstream regulatory region (URR) of the HPV types 8, 38, 92, 93 and 96, as well...... and 96 URR was up-regulated by cellular differentiation, linking the activity of these HPVs to the cellular state. UV-B irradiation activated HPV-8 but inhibited HPV-38 and HPV-93 whereas HPV-92 and 96 were not affected. As there are variable UV-B responses among the HPV types, further studies...... of interactions between UV-B and HPV need to consider the HPV type....

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

    Science.gov (United States)

    Grignard, Elise; Lapenna, Silvia; Bremer, Susanne

    2012-08-01

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

  1. Expression and Activation of STAT Transcription Factors in Breast Cancer

    Science.gov (United States)

    1998-05-08

    clinicians. J~, 273: 577-585, 1995. 183 Hundertmark 5, Buhler H, Rudolf M, Weitzel HK, Ragosch V: Inhibition of 11 beta-hydroxysteroid dehydrogenase...activated protein kinase through a Jakl-dependent pathway. Mol. Cell. Bioi., 17:3833-40, 1997. Stewart JF, Rubens RO, King RJ, Minton MJ, Steiner R

  2. Histone Acetylation Influences the Activity of Sox9-related Transcriptional Complex

    Directory of Open Access Journals (Sweden)

    Furumatsu,Takayuki

    2010-12-01

    Full Text Available Chondrocyte differentiation is the fundamental process in skeletal development. From the mesenchymal condensation of chondroprogenitors to the hypertrophic maturation of chondrocytes, chondrogenesis is sequentially regulated by cross-talk among transcription factors, growth factors, and chromatin structure. The master transcription factor Sry-type HMG box (Sox 9 has an essential role in the expression of chondrogenic genes through the association with Sox9-binding sites on its target genes. Several transcription factors and coactivators, such as Scleraxis/E47 and p300, cooperatively modulate the Sox9-dependent transcription by interacting with Sox9. The Sox9-related transcriptional apparatus activates its target gene expression through p300-mediated histone acetylation on chromatin. The transforming growth factor (TGF-β superfamily also plays a key role in chondrocyte differentiation. The TGF-β-regulated Smad3/4 complex activates Sox9-dependent transcription on chromatin by associating with Sox9 itself, and by recruiting p300 onto Sox9. These findings suggest that the epigenetic status including histone modification and chromatin structure, directly influences Sox9-regulated chondrocyte differentiation. In this article, we review the regulators of Sox9 expression itself, modulators of posttranslational Sox9 function, and Sox9-associating factors in the Sox9-dependent epigenetic regulation during chondrogenesis.

  3. Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae.

    Science.gov (United States)

    ter Schure, E G; Silljé, H H; Raeven, L J; Boonstra, J; Verkleij, A J; Verrips, C T

    1995-05-01

    Variations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.

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

    Science.gov (United States)

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

    2010-11-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Takashi Watanabe

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

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

    Science.gov (United States)

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

    2016-01-11

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

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

    Science.gov (United States)

    Lawrence, Cortney L.; Baldwin, Albert S.

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    LENUS (Irish Health Repository)

    Keld, R

    2011-06-28

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

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

    Science.gov (United States)

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

    2016-05-24

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Malik-Soni, Natasha; Frappier, Lori

    2014-02-01

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

  14. Molecular Determinants for PspA-Mediated Repression of the AAA Transcriptional Activator PspF

    OpenAIRE

    Elderkin, Sarah; Bordes, Patricia; Jones, Susan; Rappas, Mathieu; Buck, Martin

    2005-01-01

    The Escherichia coli phage shock protein system (pspABCDE operon and pspG gene) is induced by numerous stresses related to the membrane integrity state. Transcription of the psp genes requires the RNA polymerase containing the σ54 subunit and the AAA transcriptional activator PspF. PspF belongs to an atypical class of σ54 AAA activators in that it lacks an N-terminal regulatory domain and is instead negatively regulated by another regulatory protein, PspA. PspA therefore represses its own exp...

  15. Regulation of U6 Promoter Activity by Transcriptional Interference in Viral Vector-Based RNAi

    Institute of Scientific and Technical Information of China (English)

    Linghu Nie; Meghna Das Thakur; Yumei Wang; Qin Su; Yongliang Zhao; Yunfeng Feng

    2010-01-01

    The direct negative impact of the transcriptional activity of one component on the second one in c/s is referred to as transcriptional interference (TI).U6 is a type Ⅲ RNA polymerase Ⅲ promoter commonly used for driving small hairpin RNA (shRNA) expression in vector-based RNAi.In the design and construction of viral vectors,multiple transcription units may be arranged in close proximity in a space-limited vector.Determining if U6 promoter activity can be affected by TI is critical for the expression of target shRNA in gene therapy or loss-of-function studies.In this research,we designed and implemented a modified retroviral system where shRNA and exogenous gene expressions were driven by two independent transcriptional units.We arranged U6 promoter driving.shRNA expression and UbiC promoter in two promoter arrangements.In primary macrophages,we found U6 promoter activity was inhibited by UbiC promoter when in the divergent arrangement but not in tandem.In contrast,PKG promoter had no such negative impact.Instead of enhancing U6 promoter activity,CMV enhancer had significant negative impact on U6 promoter activity in the presence of UbiC promoter.Our results indicate that U6 promoter activity can be affected by TI in a proximal promoter-specific and arrangement-dependent manner.

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Model of transcriptional activation by MarA in escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-01

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

  18. Cisplatin induces cytotoxicity through the mitogen-activated protein kinase pathways and activating transcription factor 3.

    Science.gov (United States)

    St Germain, Carly; Niknejad, Nima; Ma, Laurie; Garbuio, Kyla; Hai, Tsonwin; Dimitroulakos, Jim

    2010-07-01

    The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3) as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogen-activated protein kinase (MAPK) pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38) resulted in decreased ATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/- murine embryonic fibroblasts (MEFs) were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin's cytotoxic effects.

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

    Science.gov (United States)

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

    2016-06-13

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  1. Plants contain a novel multi-member class of heat shock factors without transcriptional activator potential.

    Science.gov (United States)

    Czarnecka-Verner, E; Yuan, C X; Scharf, K D; Englich, G; Gurley, W B

    2000-07-01

    Based on phylogeny of DNA-binding domains and the organization of hydrophobic repeats, two families of heat shock transcription factors (HSFs) exist in plants. Class A HSFs are involved in the activation of the heat shock response, but the role of class B HSFs is not clear. When transcriptional activities of full-length HSFs were monitored in tobacco protoplasts, no class B HSFs from soybean or Arabidopsis showed activity under control or heat stress conditions. Additional assays confirmed the finding that the class B HSFs lacked the capacity to activate transcription. Fusion of a heterologous activation domain from human HSF1 (AD2) to the C-terminus of GmHSFB1-34 gave no evidence of synergistic enhancement of AD2 activity, which would be expected if weak activation domains were present. Furthermore, activity of AtHSFB1-4 (class B) was not rescued by coexpression with AtHSFA4-21 (class A) indicating that the class A HSF was not able to provide a missing function required for class B activity. The transcriptional activation potential of Arabidopsis AtHSFA4-21 was mapped primarily to a 39 amino acid fragment in the C-terminus enriched in bulky hydrophobic and acidic residues. Deletion mutagenesis of the C-terminal activator regions of tomato and Arabidopsis HSFs indicated that these plant HSFs lack heat-inducible regulatory regions analogous to those of mammalian HSF1. These findings suggest that heat shock regulation in plants may differ from metazoans by partitioning negative and positive functional domains onto separate HSF proteins. Class A HSFs are primarily responsible for stress-inducible activation of heat shock genes whereas some of the inert class B HSFs may be specialized for repression, or down-regulation, of the heat shock response.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sanjit K Roy

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

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

    Science.gov (United States)

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

    2013-08-01

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

  6. Inflammatory transcription factors as activation markers and functional readouts in immune-to-brain communication.

    Science.gov (United States)

    Rummel, Christoph

    2016-05-01

    Immune-to-brain communication pathways involve humoral mediators, including cytokines, central modulation by neuronal afferents and immune cell trafficking to the brain. During systemic inflammation these pathways contribute to mediating brain-controlled sickness symptoms including fever. Experimentally, activation of these signaling pathways can be mimicked and studied when injecting animals with pathogen associated molecular patterns (PAMPS). One central component of the brain inflammatory response, which leads, for example, to fever induction, is transcriptional activation of brain cells via cytokines and PAMPS. We and others have studied the spatiotemporal activation and the physiological significance of transcription factors for the induction of inflammation within the brain and the manifestation of fever. Evidence has revealed a role of nuclear factor (NF)κB in the initiation, signal transducer and activator of transcription (STAT)3 in the maintenance and NF-interleukin (IL)6 in the maintenance or even termination of brain-inflammation and fever. Moreover, psychological stressors, such as exposure to a novel environment, leads to increased body core temperature and genomic NF-IL6-activation, suggesting a potential use of NF-IL6-immunohistochemistry as a multimodal brain cell activation marker and a role for NF-IL6 for differential brain activity. In addition, the nutritional status, as reflected by circulating levels of the cytokine-like hormone leptin, influence immune-to-brain communication and age-dependent changes in LPS-induced fever. Overall, transcription factors remain therapeutically important targets for the treatment of brain-inflammation and fever induction during infectious/non-infectious inflammatory and psychological stress. However, the exact physiological role and significance of these transcription factors requires to be further investigated.

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

    Science.gov (United States)

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

    2014-10-15

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

  8. Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

    Science.gov (United States)

    Marinello, Jessica; Bertoncini, Stefania; Aloisi, Iris; Cristini, Agnese; Malagoli Tagliazucchi, Guidantonio; Forcato, Mattia; Sordet, Olivier; Capranico, Giovanni

    2016-01-01

    Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs) levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop) in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

  9. Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

    Directory of Open Access Journals (Sweden)

    Jessica Marinello

    Full Text Available Topoisomerase I-DNA-cleavage complexes (Top1cc stabilized by camptothecin (CPT have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

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

    Science.gov (United States)

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

    2015-09-11

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

  11. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    Directory of Open Access Journals (Sweden)

    Schuren Frank H

    2008-12-01

    Full Text Available Abstract Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability.

  12. Multiple transcription factor codes activate epidermal wound-response genes in Drosophila.

    Science.gov (United States)

    Pearson, Joseph C; Juarez, Michelle T; Kim, Myungjin; Drivenes, Øyvind; McGinnis, William

    2009-02-17

    Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes-Ddc, ple, msn, and kkv-that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound-response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Haiyan Hu

    2009-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Till D Frank

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

  16. Glycogen synthase kinase3 beta phosphorylates serine 33 of p53 and activates p53's transcriptional activity

    Directory of Open Access Journals (Sweden)

    Price Brendan D

    2001-07-01

    Full Text Available Abstract Background The p53 protein is activated by genotoxic stress, oncogene expression and during senescence, p53 transcriptionally activates genes involved in growth arrest and apoptosis. p53 activation is regulated by post-translational modification, including phosphorylation of the N-terminal transactivation domain. Here, we have examined how Glycogen Synthase Kinase (GSK3, a protein kinase involved in tumorigenesis, differentiation and apoptosis, phosphorylates and regulates p53. Results The 2 isoforms of GSK3, GSK3α and GSK3β, phosphorylate the sequence Ser-X-X-X-Ser(P when the C-terminal serine residue is already phosphorylated. Several p53 kinases were examined for their ability to create GSK3 phosphorylation sites on the p53 protein. Our results demonstrate that phosphorylation of serine 37 of p53 by DNA-PK creates a site for GSK3β phosphorylation at serine 33 in vitro. GSK3α did not phosphorylate p53 under any condition. GSK3β increased the transcriptional activity of the p53 protein in vivo. Mutation of either serine 33 or serine 37 of p53 to alanine blocked the ability of GSK3β to regulate p53 transcriptional activity. GSK3β is therefore able to regulate p53 function in vivo. p53's transcriptional activity is commonly increased by DNA damage. However, GSK3β kinase activity was inhibited in response to DNA damage, suggesting that GSK3β regulation of p53 is not involved in the p53-DNA damage response. Conclusions GSK3β can regulate p53's transcriptional activity by phosphorylating serine 33. However, GSK3β does not appear to be part of the p53-DNA damage response pathway. Instead, GSK3β may provide the link between p53 and non-DNA damage mechanisms for p53 activation.

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

    Science.gov (United States)

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

    2008-11-01

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

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

    Science.gov (United States)

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

    2012-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

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

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

    DEFF Research Database (Denmark)

    Koziczak, M; Müller, H; Helin, K;

    2001-01-01

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

  2. Analysis of the transcriptional networks underpinning the activation of murine macrophages by inflammatory mediators.

    Science.gov (United States)

    Raza, Sobia; Barnett, Mark W; Barnett-Itzhaki, Zohar; Amit, Ido; Hume, David A; Freeman, Tom C

    2014-08-01

    Macrophages respond to the TLR4 agonist LPS with a sequential transcriptional cascade controlled by a complex regulatory network of signaling pathways and transcription factors. At least two distinct pathways are currently known to be engaged by TLR4 and are distinguished by their dependence on the adaptor molecule MyD88. We have used gene expression microarrays to define the effects of each of three variables--LPS dose, LPS versus IFN-β and -γ, and genetic background--on the transcriptional response of mouse BMDMs. Analysis of correlation networks generated from the data has identified subnetworks or modules within the macrophage transcriptional network that are activated selectively by these variables. We have identified mouse strain-specific signatures, including a module enriched for SLE susceptibility candidates. In the modules of genes unique to different treatments, we found a module of genes induced by type-I IFN but not by LPS treatment, suggesting another layer of complexity in the LPS-TLR4 signaling feedback control. We also observe that the activation of the complement system, in common with the known activation of MHC class 2 genes, is reliant on IFN-γ signaling. Taken together, these data further highlight the exquisite nature of the regulatory systems that control macrophage activation, their likely relevance to disease resistance/susceptibility, and the appropriate response of these cells to proinflammatory stimuli.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob...

  5. Analysis of the transcriptional networks underpinning the activation of murine macrophages by inflammatory mediators

    Science.gov (United States)

    Raza, Sobia; Barnett, Mark W.; Barnett-Itzhaki, Zohar; Amit, Ido; Hume, David A.; Freeman, Tom C.

    2014-01-01

    Macrophages respond to the TLR4 agonist LPS with a sequential transcriptional cascade controlled by a complex regulatory network of signaling pathways and transcription factors. At least two distinct pathways are currently known to be engaged by TLR4 and are distinguished by their dependence on the adaptor molecule MyD88. We have used gene expression microarrays to define the effects of each of three variables—LPS dose, LPS versus IFN-β and -γ, and genetic background—on the transcriptional response of mouse BMDMs. Analysis of correlation networks generated from the data has identified subnetworks or modules within the macrophage transcriptional network that are activated selectively by these variables. We have identified mouse strain-specific signatures, including a module enriched for SLE susceptibility candidates. In the modules of genes unique to different treatments, we found a module of genes induced by type-I IFN but not by LPS treatment, suggesting another layer of complexity in the LPS-TLR4 signaling feedback control. We also observe that the activation of the complement system, in common with the known activation of MHC class 2 genes, is reliant on IFN-γ signaling. Taken together, these data further highlight the exquisite nature of the regulatory systems that control macrophage activation, their likely relevance to disease resistance/susceptibility, and the appropriate response of these cells to proinflammatory stimuli. PMID:24721704

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

    Directory of Open Access Journals (Sweden)

    Sarah To

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

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

    Science.gov (United States)

    To, Sarah; Rodda, Stephen J; Rathjen, Peter D; Keough, Rebecca A

    2010-07-22

    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.

  8. A do-it-yourself protocol for simple transcription activator-like effector assembly

    OpenAIRE

    Uhde-Stone Claudia; Gor Nilang; Chin Tiffany; Huang Joseph; Lu Biao

    2013-01-01

    Abstract Background TALEs (transcription activator-like effectors) are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, l...

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

    Science.gov (United States)

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

    2010-07-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

    Liu, Dan; Liu, Xin; Wu, Ye; Wang, Wen; Ma, Xinliang; Liu, Huirong

    2016-01-16

    HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at -1205~-838 bp and -146~+93 bp, with the -838~-649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at -709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-13

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

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

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

    Bernard, Amélie; Klionsky, Daniel J

    2015-04-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Qian-Rong; Qi; Zeng-Ming; Yang

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

  19. Cardiac hypertrophy induced by active Raf depends on Yorkie-mediated transcription

    OpenAIRE

    Yu, Lin; Daniels, Joseph P.; Wu, Huihui; Wolf, Matthew J.

    2015-01-01

    Organ hypertrophy can result from enlargement of individual cells or from cell proliferation or both. Activating mutations in the serine-threonine kinase Raf cause cardiac hypertrophy and contribute to Noonan syndrome in humans. Cardiac-specific expression of activated Raf also causes hypertrophy in Drosophila melanogaster. We found that Yorkie (Yki), a transcriptional coactivator in the Hippo pathway that regulates organ size, is required for Raf-induced cardiac hypertrophy in flies. Althoug...

  20. A central region of Gli2 regulates its localization to the primary cilium and transcriptional activity

    OpenAIRE

    Santos, Nicole; Reiter, Jeremy F.

    2014-01-01

    Signaling through vertebrate Hedgehog (Hh) proteins depends on the primary cilium. In response to Hh signals, the transcriptional activator of the pathway, Gli2, accumulates at the ciliary tip, raising the possibility that ciliary localization is important for Gli2 activation. To test this hypothesis, we used the Floxin system to create knock-in Gli2 alleles in embryonic stem cells (ESCs) to allow methodical testing of which domains and residues are essential for the ciliary localization of G...

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

    Science.gov (United States)

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

    2016-03-01

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

  2. RIT1 GTPase Regulates Sox2 Transcriptional Activity and Hippocampal Neurogenesis.

    Science.gov (United States)

    Mir, Sajad; Cai, Weikang; Andres, Douglas A

    2017-02-10

    Adult neurogenesis, the process of generating mature neurons from neuronal progenitor cells, makes critical contributions to neural circuitry and brain function in both healthy and disease states. Neurogenesis is a highly regulated process in which diverse environmental and physiological stimuli are relayed to resident neural stem cell populations to control the transcription of genes involved in self-renewal and differentiation. Understanding the molecular mechanisms governing neurogenesis is necessary for the development of translational strategies to harness this process for neuronal repair. Here we report that the Ras-related GTPase RIT1 serves to control the sequential proliferation and differentiation of adult hippocampal neural progenitor cells, with in vivo expression of active RIT1 driving robust adult neurogenesis. Gene expression profiling analysis demonstrates increased expression of a specific set of transcription factors known to govern adult neurogenesis in response to active RIT1 expression in the hippocampus, including sex-determining region Y-related HMG box 2 (Sox2), a well established regulator of stem cell self-renewal and neurogenesis. In adult hippocampal neuronal precursor cells, RIT1 controls an Akt-dependent signaling cascade, resulting in the stabilization and transcriptional activation of phosphorylated Sox2. This study supports a role for RIT1 in relaying niche-derived signals to neural/stem progenitor cells to control transcription of genes involved in self-renewal and differentiation.

  3. Yap5 is an iron-responsive transcriptional activator that regulates vacuolar iron storage in yeast.

    Science.gov (United States)

    Li, Liangtao; Bagley, Dustin; Ward, Diane M; Kaplan, Jerry

    2008-02-01

    The transporter Ccc1 imports iron into the vacuole, which is the major site of iron storage in fungi and plants. CCC1 mRNA is destabilized under low-iron conditions by the binding of Cth1 and Cth2 to the 3' untranslated region (S. Puig, E. Askeland, and D. J. Thiele, Cell 120:99-110, 2005). Here, we show that the transcription of CCC1 is stimulated by iron through a Yap consensus site in the CCC1 promoter. We identified YAP5 as being the iron-sensitive transcription factor and show that a yap5Delta strain is sensitive to high iron. Green fluorescent protein-tagged Yap5 is localized to the nucleus and occupies the CCC1 promoter independent of the iron concentration. Yap5 contains two cysteine-rich domains, and the mutation of the cysteines to alanines in each of the domains affects the transcription of CCC1 but not DNA binding. The fusion of the Yap5 cysteine-containing domains to a GAL4 DNA binding domain results in iron-sensitive GAL1-lacZ expression. Iron affects the sulfhydryl status of Yap5, which is indicative of the generation of intramolecular disulfide bonds. These results show that Yap5 is an iron-sensing transcription factor and that iron regulates transcriptional activation.

  4. Yap5 Is an Iron-Responsive Transcriptional Activator That Regulates Vacuolar Iron Storage in Yeast▿

    Science.gov (United States)

    Li, Liangtao; Bagley, Dustin; Ward, Diane M.; Kaplan, Jerry

    2008-01-01

    The transporter Ccc1 imports iron into the vacuole, which is the major site of iron storage in fungi and plants. CCC1 mRNA is destabilized under low-iron conditions by the binding of Cth1 and Cth2 to the 3′ untranslated region (S. Puig, E. Askeland, and D. J. Thiele, Cell 120:99-110, 2005). Here, we show that the transcription of CCC1 is stimulated by iron through a Yap consensus site in the CCC1 promoter. We identified YAP5 as being the iron-sensitive transcription factor and show that a yap5Δ strain is sensitive to high iron. Green fluorescent protein-tagged Yap5 is localized to the nucleus and occupies the CCC1 promoter independent of the iron concentration. Yap5 contains two cysteine-rich domains, and the mutation of the cysteines to alanines in each of the domains affects the transcription of CCC1 but not DNA binding. The fusion of the Yap5 cysteine-containing domains to a GAL4 DNA binding domain results in iron-sensitive GAL1-lacZ expression. Iron affects the sulfhydryl status of Yap5, which is indicative of the generation of intramolecular disulfide bonds. These results show that Yap5 is an iron-sensing transcription factor and that iron regulates transcriptional activation. PMID:18070921

  5. Activation of the SMU.1882 transcription by CovR in Streptococcus mutans.

    Directory of Open Access Journals (Sweden)

    Patrick Chong

    Full Text Available In Streptococcus mutans, the global response regulator CovR plays an important role in biofilm formation, stress-tolerance response, and caries production. We have previously shown that CovR acts as a transcriptional repressor by binding to the upstream promoter regions of its target genes. Here, we report that in vivo, CovR activates the transcription of SMU.1882, which encodes a small peptide containing a double-glycine motif. We also show that SMU.1882 is transcriptionally linked to comA that encodes a putative ABC transporter protein. Several genes from man gene clusters that encode mannose phosphotranferase system flank SMU.1882 -comA genes. Genomic comparison with other streptococci indicates that SMU.1882 is uniquely present in S. mutans, while the man operon is conserved among all streptococci, suggesting that a genetic rearrangement might have taken place at this locus. With the use of a transcriptional reporter system and semi-quantitative RT-PCR, we demonstrated the transcriptional regulation of SMU.1882 by CovR. In vitro gel shift and DNase I foot-printing analyses with purified CovR suggest that CovR binds to a large region surrounding the -10 region of the P(1882. Using this information and comparing with other CovR regulated promoters, we have developed a putative consensus binding sequence for CovR. Although CovR binds to P(1882, in vitro experiments using purified S. mutans RpoD, E. coli RNA polymerase, and CovR did not activate transcription from this promoter. Thus, we speculate that in vivo, CovR may interfere with the binding of a repressor or requires a cofactor.

  6. A central region of Gli2 regulates its localization to the primary cilium and transcriptional activity.

    Science.gov (United States)

    Santos, Nicole; Reiter, Jeremy F

    2014-04-01

    Signaling through vertebrate Hedgehog (Hh) proteins depends on the primary cilium. In response to Hh signals, the transcriptional activator of the pathway, Gli2, accumulates at the ciliary tip, raising the possibility that ciliary localization is important for Gli2 activation. To test this hypothesis, we used the Floxin system to create knock-in Gli2 alleles in embryonic stem cells (ESCs) to allow methodical testing of which domains and residues are essential for the ciliary localization of Gli2. The Gli2 zinc fingers, transcriptional activation domain, repressor domain, phosphorylation cluster and a Sufu binding motif were each dispensable for ciliary localization. Mutating residues that are required for Gli2 sumoylation and nuclear trafficking also did not abrogate ciliary localization. By contrast, several other domains restricted Gli2 nuclear localization, and a central region, distinct from previously characterized domains, was required for ciliary localization. In addition to an inability to localize to cilia, Gli2 lacking this central domain was unable to activate target genes. Thus, our systematic analysis in ESCs reveals that distinct regions of Gli2 regulate its nuclear and ciliary localization. The identification of a domain essential for both ciliary localization and transcriptional activity suggests that ciliary localization of Gli2 is required for its activation.

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

    Science.gov (United States)

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

    2015-01-29

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

    Science.gov (United States)

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

    2016-02-18

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

  10. Model of Transcriptional Activation By MarA in Escherichia Coli

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  13. Copper is required for cobalt-induced transcriptional activity of hypoxia-inducible factor-1.

    Science.gov (United States)

    Qiu, Liying; Ding, Xueqin; Zhang, Zhen; Kang, Y James

    2012-08-01

    Cobalt inhibits prolyl hydroxylases, leading to the accumulation of hypoxia-inducible factor-1α (HIF-1α) and a concomitant increase in the transcriptional activity of HIF-1. Therefore, cobalt has been under development as a drug for activating HIF-1 under some disease conditions. However, it has been shown that ischemic conditions resulted in the loss of copper, and the activation of HIF-1 would not occur unless copper was supplemented. The present study was undertaken to test the hypothesis that copper is also required for the cobalt activation of HIF-1 transcriptional activity. Human umbilical vein endothelial cells subjected to treatment with cobalt chloride (CoCl(2)) at concentrations above 25 μM for 2 h resulted in an accumulation of HIF-1α, which was determined by Western blot analysis, and an increase in the expression of vascular endothelial growth factor (VEGF), which was determined by real-time reverse transcription-polymerase chain reaction analysis for mRNA levels and enzyme-linked immunosorbent assay analysis for protein levels. The copper chelator tetraethylenepentamine at 25 μM did not significantly affect the accumulation of HIF-1α but blocked increases in VEGF mRNA and protein levels, an effect that could be reversed by the addition of 25 μM copper sulfate (CuSO(4)). In addition, gene silencing of the copper chaperone for Cu,Zn-superoxide dismutase blocked VEGF expression with little effect on cobalt-induced HIF-1α accumulation. The present study thus demonstrates that copper was required for cobalt-activated transcriptional activity of HIF-1, although copper did not affect cobalt-induced accumulation of HIF-1α in the cells.

  14. Bromodomain and extra-terminal (BET) bromodomain inhibition activate transcription via transient release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein.

    Science.gov (United States)

    Bartholomeeusen, Koen; Xiang, Yanhui; Fujinaga, Koh; Peterlin, B Matija

    2012-10-19

    By phosphorylating elongation factors and the C-terminal domain of RNA polymerase II, the positive transcription elongation factor b (P-TEFb) is the critical kinase for transcription elongation and co-transcriptional processing of eukaryotic genes. It exists in inactive small nuclear ribonucleoprotein (7SK snRNP) and active (free P-TEFb) complexes in cells. The P-TEFb equilibrium determines the state of cellular activation, proliferation, and differentiation. Free P-TEFb, which is required for growth, can be recruited to RNA polymerase II via transcription factors, BRD4, or the super elongation complex (SEC). UV light, various signaling cascades, transcriptional blockade, or compounds such as hexamethylene bisacetamide (HMBA), suberoylanilide hydroxamic acid (SAHA), and other histone deacetylase inhibitors lead to a rapid release of free P-TEFb, followed by its reassembly into the 7SK snRNP. As a consequence, transcription of HEXIM1, a critical 7SK snRNP subunit, and HIV is induced. In this study, we found that a bromodomain and extra-terminal (BET) bromodomain inhibitor, JQ1, which inhibits BRD4 by blocking its association with chromatin, also leads to the rapid release of free P-TEFb from the 7SK snRNP. Indeed, JQ1 transiently increased levels of free P-TEFb and BRD4·P-TEFb and SEC·P-TEFb complexes in cells. As a consequence, the levels of HEXIM1 and HIV proteins rose. Importantly, the knockdown of ELL2, a subunit of the SEC, blocked the ability of JQ1 to increase HIV transcription. Finally, the effects of JQ1 and HMBA or SAHA on the P-TEFb equilibrium were cooperative. We conclude that HMBA, SAHA, and JQ1 affect transcription elongation by a similar and convergent mechanism.

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

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    Qinghe Chen

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

  16. Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

    Directory of Open Access Journals (Sweden)

    Carly St. Germain

    2010-07-01

    Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

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

    Science.gov (United States)

    Kang, S

    1993-08-25

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

  18. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Iordanskiy, Sergey [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Van Duyne, Rachel [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Romerio, Fabio [Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Kashanchi, Fatah, E-mail: fkashanc@gmu.edu [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States)

    2015-11-15

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation

  19. Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration.

    Science.gov (United States)

    Martin, Robert G; Bartlett, Emily S; Rosner, Judah L; Wall, Michael E

    2008-07-04

    The paralogous transcriptional activators MarA, SoxS, and Rob activate a common set of promoters, the marA/soxS/rob regulon of Escherichia coli, by binding a cognate site (marbox) upstream of each promoter. The extent of activation varies from one promoter to another and is only poorly correlated with the in vitro affinity of the activator for the specific marbox. Here, we examine the dependence of promoter activation on the level of activator in vivo by manipulating the steady-state concentrations of MarA and SoxS in Lon protease mutants and by measuring promoter activation using lacZ transcriptional fusions. We found that: (i) the MarA concentrations needed for half-maximal stimulation varied by at least 19-fold among the 10 promoters tested; (ii) most marboxes were not saturated when there were 24,000 molecules of MarA per cell; (iii) the correlation between the MarA concentration needed for half-maximal promoter activity in vivo and marbox binding affinity in vitro was poor; and (iv) the two activators differed in their promoter activation profiles. The marRAB and sodA promoters could both be saturated by MarA and SoxS in vivo. However, saturation by MarA resulted in greater marRAB and lesser sodA transcription than did saturation by SoxS, implying that the two activators interact with RNA polymerase in different ways at the different promoters. Thus, the concentration and nature of activator determine which regulon promoters are activated, as well as the extent of their activation.

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

    Science.gov (United States)

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

    2016-01-01

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

  1. IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity.

    Science.gov (United States)

    Sayedyahossein, Samar; Li, Zhigang; Hedman, Andrew C; Morgan, Chase J; Sacks, David B

    2016-09-09

    During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2014-01-13

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

  4. A co-activator of nitrogen-regulated transcription in Saccharomyces cerevisiae.

    Science.gov (United States)

    Soussi-Boudekou, S; André, B

    1999-02-01

    In Saccharomyces cerevisiae, the transcription factors Gln3p and Nil1p of the GATA family play a determinant role in expression of genes that are subject to nitrogen catabolite repression. Here we report the isolation of a new yeast mutant, gan1-1, exhibiting dramatically decreased NAD-linked glutamate dehydrogenase (NAD-GDH) and glutamine synthetase (GS) activities. The GAN1 gene was cloned and found to encode a 488-amino-acid polypeptide bearing no typical DNA binding domain. Gan1p is required for full expression of GLN1, GDH2 and also other nitrogen utilization genes, including GAP1, PUT4, MEP2 and GDH1. The extent to which Gan1p is required, however, varies according to the gene and to the nitrogen source available. We show that Gan1p is in fact involved in Gln3p- and Nil1p-dependent transcription. In the case of Gln3p-dependent transcription, the degree to which Gan1p is required appears to be gene specific. The contribution of Gan1p to gene expression is also influenced by the nitrogen status of the cell. We found that GAN1 is identical to ADA1, which encodes a component of the ADA/GCN5 co-activator complex. Ada1/Gan1p thus represents the first reported case of an accessory protein (a co-activator) linking the GATA-binding proteins Gln3p and Nil1p, mediating nitrogen-regulated transcription, to the basal transcription machinery.

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

    Science.gov (United States)

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

    2014-02-13

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

  6. O-GlcNAc modification of PPAR{gamma} reduces its transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Suena; Park, Sang Yoon [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Roth, Juergen [Department of Integrated OMICS for Biomedical Science, Graduate School, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Hoe Suk, E-mail: hoeskim@snu.ac.kr [Institute of Radiation Medicine, Medical Research Center, Seoul National University, Seoul 110-744 (Korea, Republic of); Cho, Jin Won, E-mail: chojw311@yonsei.ac.kr [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Department of Integrated OMICS for Biomedical Science, Graduate School, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer We found that PPAR{gamma} is modified by O-GlcNAc in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer The Thr54 of PPAR{gamma}1 is the major O-GlcNAc site. Black-Right-Pointing-Pointer Transcriptional activity of PPAR{gamma}1 was decreased on treatment with the OGA inhibitor. -- Abstract: The peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear receptor superfamily, is a key regulator of adipogenesis and is important for the homeostasis of the adipose tissue. The {beta}-O-linked N-acetylglucosamine (O-GlcNAc) modification, a posttranslational modification on various nuclear and cytoplasmic proteins, is involved in the regulation of protein function. Here, we report that PPAR{gamma} is modified by O-GlcNAc in 3T3-L1 adipocytes. Mass spectrometric analysis and mutant studies revealed that the threonine 54 of the N-terminal AF-1 domain of PPAR{gamma} is the major O-GlcNAc site. Transcriptional activity of wild type PPAR{gamma} was decreased 30% by treatment with the specific O-GlcNAcase (OGA) inhibitor, but the T54A mutant of PPAR{gamma} did not respond to inhibitor treatment. In 3T3-L1 cells, an increase in O-GlcNAc modification by OGA inhibitor reduced PPAR{gamma} transcriptional activity and terminal adipocyte differentiation. Our results suggest that the O-GlcNAc state of PPAR{gamma} influences its transcriptional activity and is involved in adipocyte differentiation.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-08

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

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

    Directory of Open Access Journals (Sweden)

    Yu Deng

    2013-01-01

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

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

    Science.gov (United States)

    Payton-Stewart, Florastina; Tilghman, Syreeta L; Williams, LaKeisha G; Winfield, Leyte L

    2014-08-08

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

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

    Directory of Open Access Journals (Sweden)

    José Perdomo

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

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

    Science.gov (United States)

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

    2013-04-01

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

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

    Directory of Open Access Journals (Sweden)

    V.S. Carrara

    2009-06-01

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

  13. Identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the RNA polymerase.

    Science.gov (United States)

    Nuez, B; Rojo, F; Barthelemy, I; Salas, M

    1991-05-11

    Expression of Bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4. This activator binds to a region of the late A3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending Tin the DNA. In this work the target sequences recognized by protein p4 in the phage phi 29 late A3 promoter have been characterized. The binding of protein p4 to derivatives of the late A3 promoter harbouring deletions in the protein p4 binding site has been studied. When protein p4 recognition sequences were altered, the activator could only bind to the promoter in the presence of RNA polymerase. This strong cooperativity in the binding of protein p4 and RNA polymerase to the promoter suggests the presence of direct protein-protein contacts between them.

  14. Transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in Bacillus subtilis.

    Science.gov (United States)

    Fedorova, Ksenia; Kayumov, Airat; Woyda, Kathrin; Ilinskaja, Olga; Forchhammer, Karl

    2013-05-02

    The Bacillus subtilis glutamine synthetase (GS) plays a dual role in cell metabolism by functioning as catalyst and regulator. GS catalyses the ATP-dependent synthesis of glutamine from glutamate and ammonium. Under nitrogen-rich conditions, GS becomes feedback-inhibited by high intracellular glutamine levels and then binds transcription factors GlnR and TnrA, which control the genes of nitrogen assimilation. While GS-bound TnrA is no longer able to interact with DNA, GlnR-DNA binding is shown to be stimulated by GS complex formation. In this paper we show a new physiological feature of the interaction between glutamine synthetase and TnrA. The transcription factor TnrA inhibits the biosynthetic activity of glutamine synthetase in vivo and in vitro, while the GlnR protein does not affect the activity of the enzyme.

  15. Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters.

    Science.gov (United States)

    Beaucage, S L; Miller, C A; Cohen, S N

    1991-09-01

    The pSC101 plasmid encodes a cis-acting genetic locus termed par that ensures the stable inheritance of plasmids in a population of dividing cells. In the absence of selection, par-defective plasmids are lost rapidly from the bacterial population. We report here that the stability of par-deleted pSC101 derivatives is restored by introducing certain adventitious bacterial promoters onto the plasmid. Stabilization requires active transcription from the inserted promoter and is affected by the site and orientation of the insertion, the length of the nascent transcript and DNA gyrase activity. While a promotor-associated overall increase in negative superhelicity of plasmid DNA was observed, stabilized inheritance appeared to be dependent on localized rather than generalized supercoiling. Our demonstration that promoter-induced DNA supercoiling can mimic the effects of the pSC101 par locus provides evidence that the previously reported superhelicity-generating effects of par are intrinsic to its function.

  16. Generation of knockout rabbits using transcription activator-like effector nucleases.

    Science.gov (United States)

    Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

    2014-01-01

    Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  17. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks.

    Science.gov (United States)

    Aymard, François; Bugler, Beatrix; Schmidt, Christine K; Guillou, Emmanuelle; Caron, Pierre; Briois, Sébastien; Iacovoni, Jason S; Daburon, Virginie; Miller, Kyle M; Jackson, Stephen P; Legube, Gaëlle

    2014-04-01

    Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

  18. Cyclin C regulates adipogenesis by stimulating transcriptional activity of CCAAT/enhancer binding protein alpha.

    Science.gov (United States)

    Song, Ziyi; Xiaoli, Alus M; Zhang, Quanwei; Zhang, Yi; Yang, Ellen S T; Wang, Sven; Chang, Rui; Zhang, Zhengdong D; Yang, Gongshe; Strich, Randy; Pessin, Jeffrey E; Yang, Fajun

    2017-03-28

    Brown adipose tissue (BAT) is important for maintaining energy homeostasis and adaptive thermogenesis in rodents and humans. As disorders arising from dysregulated energy metabolism, such as obesity and metabolic diseases, have increased, so has interest in the molecular mechanisms in adipocyte biology. Using a functional screen, we identified cyclin C (CycC), a conserved subunit of the Mediator complex, as a novel regulator for brown adipocyte formation. siRNA-mediated CycC knockdown (KD) in brown preadipocytes impaired the early transcriptional program of differentiation, and genetic knockout (KO) of CycC completely blocked the differentiation process. RNA-seq analyses of CycC-KD revealed a critical role of CycC in activating genes co-regulated by peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein alpha (C/EBPα). Overexpression of PPARγ2 or addition of the PPARγ ligand rosiglitazone rescued the defects in CycC-KO brown preadipocytes, and efficiently activated the PPARγ-responsive promoters in both wild-type (WT) and CycC-KO cells, suggesting that CycC is not essential for PPARγ transcriptional activity. In contrast, CycC-KO significantly reduced C/EBPα-dependent gene expression. Unlike for PPARγ, overexpression of C/EBPα could not induce C/EBPα target gene expression in CycC-KO cells or rescue the CycC-KO defects in brown adipogenesis, suggesting that CycC is essential for C/EBPα-mediated gene activation. CycC physically interacted with C/EBPα and this interaction was required for C/EBPα transactivation domain activity. Consistent with the role of C/EBPα in white adipogenesis, CycC-KD also inhibited differentiation of 3T3-L1 cells into white adipocytes. Together, these data indicate that CycC activates adipogenesis by stimulating the transcriptional activity of C/EBPα.

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

    Science.gov (United States)

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

    2012-03-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

  1. Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs)

    OpenAIRE

    Moore, Finola E.; Deepak Reyon; Sander, Jeffry D.; Sarah A Martinez; Blackburn, Jessica S; Cyd Khayter; Ramirez, Cherie L.; J Keith Joung; Langenau, David M.

    2012-01-01

    Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebra...

  2. Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

    OpenAIRE

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This in...

  3. Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos

    OpenAIRE

    Lei, Yong; Guo, Xiaogang; Deng, Yi; Chen, Yonglong; Zhao, Hui

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embr...

  4. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    OpenAIRE

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-01-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding reg...

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Zhang Xifeng

    2010-09-01

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

  7. Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin

    OpenAIRE

    Kakarougkas, Andreas; Ismail, Amani; Chambers, Anna; Riballo, Queti; Herbert, Alex; Kunzel, Julia; Lobrich, Markus; Jeggo, Penny; Downs, Jessica

    2014-01-01

    Summary Actively transcribed regions of the genome are vulnerable to genomic instability. Recently, it was discovered that transcription is repressed in response to neighboring DNA double-strand breaks (DSBs). It is not known whether a failure to silence transcription flanking DSBs has any impact on DNA repair efficiency or whether chromatin remodelers contribute to the process. Here, we show that the PBAF remodeling complex is important for DSB-induced transcriptional silencing and promotes ...

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

    Directory of Open Access Journals (Sweden)

    Florence P. Varodayan

    2013-12-01

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

  9. Solasonine, A Natural Glycoalkaloid Compound, Inhibits Gli-Mediated Transcriptional Activity

    Directory of Open Access Journals (Sweden)

    Jun Yang

    2016-10-01

    Full Text Available The major obstacle limiting the efficacy of current Smoothened (Smo inhibitors is the primary and acquired resistance mainly caused by Smo mutations and Gli amplification. In this context, developing Hh inhibitors targeting Gli, the final effector of this signaling pathway, may combat the resistance. In this study we found that solasonine, a natural glycoalkaloid compound, significantly inhibited the hedgehog (Hh pathway activity. Meanwhile, solasonine may obviously inhibit the alkaline phosphatase (ALP activity in C3H10T1/2 cells, concomitantly with reductions of the mRNA expression of Gli1 and Ptch1. However, we found that solasonine exhibited no effect on the transcriptional factors activities provoked by TNF-α and PGE2, thus suggesting its selectivity against Hh pathway activity. Furthermore, we identified that solasonine inhibited the Hh pathway activity by acting on its transcriptional factor Gli using a series of complementary data. We also observed that solasonine obviously inhibited the Gli-luciferase activity provoked by ectopic expression of Smo mutants which may cause the resistance to the current Smo inhibitors. Our study suggests that solasonine may significantly inhibit the Hh pathway activity by acting on Gli, therefore indicating the possibility to use solasonine as a lead compound to develop anticancer drugs for combating the resistance of current Smo inhibitors.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-04-01

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

  12. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    Science.gov (United States)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

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

    Directory of Open Access Journals (Sweden)

    Helena Bujalka

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-26

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

  15. Cardiac hypertrophy induced by active Raf depends on Yorkie-mediated transcription.

    Science.gov (United States)

    Yu, Lin; Daniels, Joseph P; Wu, Huihui; Wolf, Matthew J

    2015-02-03

    Organ hypertrophy can result from enlargement of individual cells or from cell proliferation or both. Activating mutations in the serine-threonine kinase Raf cause cardiac hypertrophy and contribute to Noonan syndrome in humans. Cardiac-specific expression of activated Raf also causes hypertrophy in Drosophila melanogaster. We found that Yorkie (Yki), a transcriptional coactivator in the Hippo pathway that regulates organ size, is required for Raf-induced cardiac hypertrophy in flies. Although aberrant activation of Yki orthologs stimulates cardiac hyperplasia in mice, cardiac-specific expression of an activated mutant form of Yki in fruit flies caused cardiac hypertrophy without hyperplasia. Knockdown of Yki caused cardiac dilation without loss of cardiomyocytes and prevented Raf-induced cardiac hypertrophy. In flies, Yki-induced cardiac hypertrophy required the TEA domain-containing transcription factor Scalloped, and, in mammalian cells, expression of mouse Raf(L613V), an activated form of Raf with a Noonan syndrome mutation, increased Yki-induced Scalloped activity. Furthermore, overexpression of Tgi (a Tondu domain-containing Scalloped-binding corepressor) in the fly heart abrogated Yki- or Raf-induced cardiac hypertrophy. Thus, crosstalk between Raf and Yki occurs in the heart and can influence Raf-mediated cardiac hypertrophy.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-18

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

  17. Activation of the Ig Iα1 promoter by the transcription factor Ets-1 triggers Ig Iα1-Cα1 germline transcription in epithelial cancer cells.

    Science.gov (United States)

    Duan, Zhi; Zheng, Hui; Xu, San; Jiang, Yiqun; Liu, Haidan; Li, Ming; Hu, Duosha; Li, Wei; Bode, Ann M; Dong, Zigang; Cao, Ya

    2014-03-01

    Immunoglobulins (Igs) are known to be synthesized and secreted only by B lymphocytes. Class switch recombination (CSR) is a key event that enables B cells to express Igs, and one of the crucial steps for CSR initiation is the germline transcription of Ig genes. Surprisingly, recent studies have demonstrated that the Ig genes are also expressed in some epithelial cancer cells; however, the mechanisms underlying how cancer cells initiate CSR and express Igs are still unknown. In this study, we confirmed that the Ig Iα1 promoter in cancer cell lines was activated by the Ets-1 transcription factor, and the activity of the Ig Iα1 promoter and Ig Iα1-Cα1 germline transcription were attenuated after knockdown of Ets-1 by specific small interfering RNAs (siRNA). Furthermore, the expression of Ets-1 and Igα heavy chain in cancer cells was dose dependently upregulated by TGF-β1. These results indicate that activation of the Ig Iα1 promoter by the transcription factor Ets-1 is a critical pathway and provides a novel mechanism for Ig expression in non-B cell cancers.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition......, Ad 0.2 µg/ml; total transcriptional inhibition, AD 2.0 µg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 µg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy...

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

    Science.gov (United States)

    Heo, Sun-Hee; Cho, Je-Yoel

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-27

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

  1. Structural Analysis of the Phenol-Responsive Sensory Domain of the Transcription Activator PoxR.

    Science.gov (United States)

    Patil, Vinod Vikas; Park, Kwang-Hyun; Lee, Seung-Goo; Woo, Euijeon

    2016-04-05

    Positive phenol-degradative gene regulator (PoxR) is a σ(54)-dependent AAA+ ATPase transcription activator that regulates the catabolism of phenols. The PoxR sensory domain detects phenols and relays signals for the activation of transcription. Here we report the first structure of the phenol sensory domain bound to phenol and five derivatives. It exists as a tightly intertwined homodimer with a phenol-binding pocket buried inside, placing two C termini on the same side of the dimer. His102 and Trp130 interact with the hydroxyl group of the phenol in a cavity surrounded by rigid hydrophobic residues on one side and a flexible region on the other. Each monomer has a V4R fold with a unique zinc-binding site. A shift at the C-terminal helix suggests that there is a possible conformational change upon ligand binding. The results provide a structural basis of chemical effector binding for transcriptional regulation with broad implications for protein engineering.

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

    Directory of Open Access Journals (Sweden)

    Shadi Jafari

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

  3. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element.

    Directory of Open Access Journals (Sweden)

    Frederick M Stanley

    Full Text Available Plasminogen activator inhibitor type 1 (PAI-1 is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter.

  4. Genetic and physiological activation of osmosensitive gene expression mimics transcriptional signatures of pathogen infection in C. elegans.

    Directory of Open Access Journals (Sweden)

    Anne-Katrin Rohlfing

    Full Text Available The soil-dwelling nematode C. elegans is a powerful system for comparative molecular analyses of environmental stress response mechanisms. Infection of worms with bacterial and fungal pathogens causes the activation of well-characterized innate immune transcriptional programs in pathogen-exposed hypodermal and intestinal tissues. However, the pathophysiological events that drive such transcriptional responses are not understood. Here, we show that infection-activated transcriptional responses are, in large part, recapitulated by either physiological or genetic activation of the osmotic stress response. Microarray profiling of wild type worms exposed to non-lethal hypertonicity identified a suite of genes that were also regulated by infection. Expression profiles of five different osmotic stress resistant (osr mutants under isotonic conditions reiterated the wild type transcriptional response to osmotic stress and also showed substantial similarity to infection-induced gene expression under isotonic conditions. Computational, transgenic, and functional approaches revealed that two GATA transcription factors previously implicated in infection-induced transcriptional responses, elt-2 and elt-3, are also essential for coordinated tissue-specific activation of osmosensitive gene expression and promote survival under osmotically stressful conditions. Together, our data suggest infection and osmotic adaptation share previously unappreciated transcriptional similarities which might be controlled via regulation of tissue-specific GATA transcription factors.

  5. Identification of transcriptional activators for thienamycin and cephamycin C biosynthetic genes within the thienamycin gene cluster from Streptomyces cattleya.

    Science.gov (United States)

    Rodríguez, Miriam; Núñez, Luz Elena; Braña, Alfredo F; Méndez, Carmen; Salas, José A; Blanco, Gloria

    2008-08-01

    Two regulatory genes, thnI and thnU, were identified in the thienamycin (thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished thienamycin production showing its involvement in thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for thienamycin biosynthesis that regulates the expression of nine genes involved in thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.

  6. Calmodulin-binding transcription activator (CAMTA) 3 mediates biotic defense responses in Arabidopsis.

    Science.gov (United States)

    Galon, Yael; Nave, Roy; Boyce, Joy M; Nachmias, Dikla; Knight, Marc R; Fromm, Hillel

    2008-03-19

    Calmodulin-binding transcription activator (CAMTA) 3 (also called SR1) is a calmodulin-binding transcription factor in Arabidopsis. Two homozygous T-DNA insertion mutants (camta3-1, camta3-2) showed enhanced spontaneous lesions. Transcriptome analysis of both mutants revealed 6 genes with attenuated expression and 99 genes with elevated expression. Of the latter, 32 genes are related to defense against pathogens (e.g. WRKY33, PR1 and chitinase). Propagation of a virulent strain of the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea were attenuated in both mutants. Moreover, both mutants accumulated high levels of H2O2. We suggest that CAMTA3 regulates the expression of a set of genes involved in biotic defense responses.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-01-01

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

  8. Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae.

    Science.gov (United States)

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

    2015-01-01

    In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5'-ATTTGAACATTATTCAAGT-3') in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.

  9. Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).

    Science.gov (United States)

    Lei, Yong; Guo, Xiaogang; Liu, Yun; Cao, Yang; Deng, Yi; Chen, Xiongfeng; Cheng, Christopher H K; Dawid, Igor B; Chen, Yonglong; Zhao, Hui

    2012-10-23

    Transcription activator-like effector nucleases (TALENs) are an approach for directed gene disruption and have been proved to be effective in various animal models. Here, we report that TALENs can induce somatic mutations in Xenopus embryos with reliably high efficiency and that such mutations are heritable through germ-line transmission. We modified the Golden Gate method for TALEN assembly to make the product suitable for RNA transcription and microinjection into Xenopus embryos. Eight pairs of TALENs were constructed to target eight Xenopus genes, and all resulted in indel mutations with high efficiencies of up to 95.7% at the targeted loci. Furthermore, mutations induced by TALENs were highly efficiently passed through the germ line to F(1) frogs. Together with simple and reliable PCR-based approaches for detecting TALEN-induced mutations, our results indicate that TALENs are an effective tool for targeted gene editing/knockout in Xenopus.

  10. CDPK1, a calcium-dependent protein kinase, regulates transcriptional activator RSG in response to gibberellins.

    Science.gov (United States)

    Nakata, Masaru; Yuasa, Takashi; Takahashi, Yohsuke; Ishida, Sarahmi

    2009-05-01

    The homeostasis of gibberellins (GAs) is maintained by negative-feedback regulation in plant cells. REPRESSION OF SHOOT GROWTH (RSG) is a transcriptional activator with a basic Leu zipper domain suggested to contribute GA feedback regulation by the transcriptional regulation of genes encoding GA biosynthetic enzymes. The 14-3-3 signaling proteins negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The phosphorylation on Ser-114 of RSG is essential for 14-3-3 binding of RSG; however, the kinase that catalyzes the reaction is unknown. Recently a Ca(2+)-dependent protein kinase (CDPK) was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of the Ser-114 of RSG. Our results suggest that CDPK decodes the Ca(2+) signal produced by GAs and regulates the intracellular localization of RSG in plant cells.

  11. Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Neff, Michael M.

    2011-06-23

    This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

  12. FOXR2 Interacts with MYC to Promote Its Transcriptional Activities and Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Xu Li

    2016-07-01

    Full Text Available By combining the results of a large-scale proteomic analysis of the human transcription factor interaction network with knowledge databases, we identified FOXR2 as one of the top-ranked candidate proto-oncogenes. Here, we show that FOXR2 forms a stable complex with MYC and MAX and subsequently regulates cell proliferation by promoting MYC’s transcriptional activities. We demonstrate that FOXR2 is highly expressed in several breast, lung, and liver cancer cell lines and related patient tumor samples, while reduction of FOXR2 expression in a xenograft model inhibits tumor growth. These results indicate that FOXR2 acts with MYC to promote cancer cell proliferation, which is a potential tumor-specific target for therapeutic intervention against MYC-driven cancers.

  13. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

    Energy Technology Data Exchange (ETDEWEB)

    Matt Vaughn Greg Harrington Daniel R Bush

    2002-08-06

    This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose

  14. In Vitro Anticancer Activity of Phlorofucofuroeckol A via Upregulation of Activating Transcription Factor 3 against Human Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Hyun Ji Eo

    2016-03-01

    Full Text Available Phlorofucofuroeckol A (PFF-A, one of the phlorotannins found in brown algae, has been reported to exert anti-cancer property. However, the molecular mechanism for the anti-cancer effect of PFF-A has not been known. Activating transcription factor 3 (ATF3 has been reported to be associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which PFF-A stimulates ATF3 expression and apoptosis in human colorectal cancer cells. PFF-A decreased cell viability through apoptosis of human colorectal cancer cells. PFF-A increased ATF3 expression through regulating transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by PFF-A was cAMP response element binding protein (CREB, located between positions −147 and −85 of the ATF3 promoter. Inhibition of p38, c-Jun N-terminal kinases (JNK, glycogen synthase kinase (GSK 3β, and IκB kinase (IKK-α blocked PFF-A-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of poly (ADP-ribose polymerase (PARP by PFF-A, while ATF3 overexpression increased PFF-A-mediated cleaved PARP. These results suggest that PFF-A may exert anti-cancer property through inducing apoptosis via the ATF3-mediated pathway in human colorectal cancer cells.

  15. Genetic variants in ABCA1 promoter affect transcription activity and plasma HDL level in pigs.

    Science.gov (United States)

    Dang, Xiao-yong; Chu, Wei-wei; Shi, Heng-chuan; Yu, Shi-gang; Han, Hai-yin; Gu, Shu-Hua; Chen, Jie

    2015-01-25

    Excess accumulation of cholesterol in plasma may result in coronary artery disease. Numerous studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for plasma high density lipoprotein (HDL) formation. Higher plasma levels of HDL are associated with lower risk for cardiovascular disease. Studies of human disease and animal models had shown that an increased hepatic ABCA1 activity relates to an enhanced plasma HDL level. In this study, we hypothesized that functional mutations in the ABCA1 promoter in pigs may affect gene transcription activity, and consequently the HDL level in plasma. The promoter region of ABCA1 was comparatively scanned by direct sequencing with pool DNA of high- and low-HDL groups (n=30 for each group). Two polymorphisms, c. - 608A>G and c. - 418T>A, were revealed with reverse allele distribution in the two groups. The two polymorphisms were completely linked and formed only G-A or A-T haplotypes when genotyped in a larger population (n=526). Furthermore, we found that the G-A/G-A genotype was associated with higher HDL and ABCA1 mRNA level than A-T/A-T genotype. Luciferase assay also revealed that G-A haplotype promoter had higher activity than A-T haplotype. Single-nucleotide mutant assay showed that c.-418T>A was the causal mutation for ABCA1 transcription activity alteration. Conclusively, we identified two completely linked SNPs in porcine ABCA1 promoter region which have influence on the plasma HDL level by altering ABCA1 gene transcriptional activity.

  16. Bnip3 Binds and Activates p300: Possible Role in Cardiac Transcription and Myocyte Morphology.

    Directory of Open Access Journals (Sweden)

    John W Thompson

    Full Text Available Bnip3 is a hypoxia-regulated member of the Bcl-2 family of proteins that is implicated in apoptosis, programmed necrosis, autophagy and mitophagy. Mitochondria are thought to be the primary targets of Bnip3 although its activities may extend to the ER, cytoplasm, and nucleus. Bnip3 is induced in the heart by ischemia and pressure-overload, and may contribute to cardiomyopathy and heart failure. Only mitochondrial-dependent programmed death actions have been described for Bnip3 in the heart. Here we describe a novel activity of Bnip3 in cultured cardiac myocytes and transgenic mice overexpressing Bnip3 in the heart (Bnip3-TG. In cultured myocytes Bnip3 bound and activated the acetyltransferase p300, increased acetylation of histones and the transcription factor GATA4, and conferred p300 and GATA4-sensitive cellular morphological changes. In intact Bnip3-TG hearts Bnip3 also bound p300 and GATA4 and conferred enhanced GATA4 acetylation. Bnip3-TG mice underwent age-dependent ventricular dilation and heart failure that was partially prevented by p300 inhibition with curcumin. The results suggest that Bnip3 regulates cardiac gene expression and perhaps myocyte morphology by activating nuclear p300 acetyltransferase activity and hyperacetylating histones and p300-selective transcription factors.

  17. The Escherichia coli RNA polymerase alpha subunit and transcriptional activation by bacteriophage lambda CII protein.

    Science.gov (United States)

    Gabig, M; Obuchowski, M; Ciesielska, A; Latała, B; Wegrzyn, A; Thomas, M S; Wegrzyn, G

    1998-01-01

    Bacteriophage lambda is not able to lysogenise the Escherichia coli rpoA341 mutant. This mutation causes a single amino acid substitution Lys271Glu in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Our previous studies indicated that the impaired lysogenisation of the rpoA341 host is due to a defect in transcriptional activation by the phage CII protein and suggested a role for alphaCTD in this process. Here we used a series of truncation and point mutants in the rpoA gene placed on a plasmid to investigate the process of transcriptional activation by the cII gene product. Our results indicate that amino-acid residues 265, 268 and 271 in the a subunit may play an important role in the CII-mediated activation of the pE promoter (most probably residue 271) or may be involved in putative interactions between alphaCTD and an UP-like element near pE (most probably residues 265 and 268). Measurement of the activity of pE-lacZ, pI-lacZ and p(aQ)-lacZ fusions in the rpoA+ and rpoA341 hosts demonstrated that the mechanism of activation of these CII-dependent promoters may be in each case different.

  18. A trihelix DNA binding protein counterbalances hypoxia-responsive transcriptional activation in Arabidopsis.

    Science.gov (United States)

    Giuntoli, Beatrice; Lee, Seung Cho; Licausi, Francesco; Kosmacz, Monika; Oosumi, Teruko; van Dongen, Joost T; Bailey-Serres, Julia; Perata, Pierdomenico

    2014-09-01

    Transcriptional activation in response to hypoxia in plants is orchestrated by ethylene-responsive factor group VII (ERF-VII) transcription factors, which are stable during hypoxia but destabilized during normoxia through their targeting to the N-end rule pathway of selective proteolysis. Whereas the conditionally expressed ERF-VII genes enable effective flooding survival strategies in rice, the constitutive accumulation of N-end-rule-insensitive versions of the Arabidopsis thaliana ERF-VII factor RAP2.12 is maladaptive. This suggests that transcriptional activation under hypoxia that leads to anaerobic metabolism may need to be fine-tuned. However, it is presently unknown whether a counterbalance of RAP2.12 exists. Genome-wide transcriptome analyses identified an uncharacterized trihelix transcription factor gene, which we named HYPOXIA RESPONSE ATTENUATOR1 (HRA1), as highly up-regulated by hypoxia. HRA1 counteracts the induction of core low oxygen-responsive genes and transcriptional activation of hypoxia-responsive promoters by RAP2.12. By yeast-two-hybrid assays and chromatin immunoprecipitation we demonstrated that HRA1 interacts with the RAP2.12 protein but with only a few genomic DNA regions from hypoxia-regulated genes, indicating that HRA1 modulates RAP2.12 through protein-protein interaction. Comparison of the low oxygen response of tissues characterized by different levels of metabolic hypoxia (i.e., the shoot apical zone versus mature rosette leaves) revealed that the antagonistic interplay between RAP2.12 and HRA1 enables a flexible response to fluctuating hypoxia and is of importance to stress survival. In Arabidopsis, an effective low oxygen-sensing response requires RAP2.12 stabilization followed by HRA1 induction to modulate the extent of the anaerobic response by negative feedback regulation of RAP2.12. This mechanism is crucial for plant survival under suboptimal oxygenation conditions. The discovery of the feedback loop regulating the oxygen

  19. Effects of calmodulin on DNA-binding activity of heat shock transcription factor in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

  20. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  1. An inducible transcription factor activates expression of human immunodeficiency virus in T cells

    Science.gov (United States)

    Nabel, Gary; Baltimore, David

    1987-04-01

    Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines1,2. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III (refs 3-6) and art genes7. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-κB (ref. 8), with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-κB acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).

  2. Gas6/Axl pathway promotes tumor invasion through the transcriptional activation of Slug in hepatocellular carcinoma.

    Science.gov (United States)

    Lee, Hsin-Jung; Jeng, Yung-Ming; Chen, Yu-Ling; Chung, Ling; Yuan, Ray-Hwang

    2014-04-01

    Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide. Other than the sorafenib treatment, no effective systemic therapy has been available thus far. Most targets in molecularly targeted therapy for cancer are receptor tyrosine kinases (RTKs). Therefore, identifying activated RTKs in HCC is critical for developing new molecularly targeted therapies. Using a phospho-RTK array, we found that Axl is one of the most frequently activated RTKs in liver cancer cell lines. The knockdown of Axl by RNA interference significantly reduced cell migration and invasion in the HCC cell lines HA22T and Mahlavu. Stimulation of HCC cell lines by Axl ligand growth arrest-specific 6 (Gas6) enhanced cell migration and invasion. The Gas6/Axl pathway enhanced the expression of the epithelial-mesenchymal transition-inducing transcription factor Slug, which is essential for the invasion-promoting activity of Axl. Treating HCC cells with the Axl inhibitor bosutinib suppressed Slug expression and decreased the invasiveness of HCC cell lines. These findings indicate that Gas6/Axl regulates tumor invasion through the transcriptional activation of Slug.

  3. Sumoylation regulates the transcriptional activity of MafA in pancreatic beta cells.

    Science.gov (United States)

    Shao, Chunli; Cobb, Melanie H

    2009-01-30

    MafA is a transcriptional regulator expressed primarily in pancreatic beta cells. It binds to the RIPE3b/C1-binding site within the ins gene promoter, which plays a critical role in regulating ins gene expression in response to glucose. Here, we show that MafA is post-translationally modified by the small ubiquitin-related modifiers SUMO-1 and -2. Mutation of a single site in MafA, Lys(32), blocks its sumoylation in beta cells. Incubation of beta cells in low glucose (2 mm) or exposure to hydrogen peroxide increases sumoylation of endogenous MafA. Forced sumoylation of MafA results in reduced transcriptional activity toward the ins gene promoter and increased suppression of the CHOP-10 gene promoter. Sumoylation of MafA has no apparent effect on either its nuclear localization in beta cells or its ubiquitin-dependent degradation. This study suggests that modification of MafA by SUMO modulates gene transcription and thereby beta cell function.

  4. Stathmin regulates mutant p53 stability and transcriptional activity in ovarian cancer.

    Science.gov (United States)

    Sonego, Maura; Schiappacassi, Monica; Lovisa, Sara; Dall'Acqua, Alessandra; Bagnoli, Marina; Lovat, Francesca; Libra, Massimo; D'Andrea, Sara; Canzonieri, Vincenzo; Militello, Loredana; Napoli, Marco; Giorda, Giorgio; Pivetta, Barbara; Mezzanzanica, Delia; Barbareschi, Mattia; Valeri, Barbara; Canevari, Silvana; Colombatti, Alfonso; Belletti, Barbara; Del Sal, Giannino; Baldassarre, Gustavo

    2013-05-01

    Stathmin is a p53-target gene, frequently overexpressed in late stages of human cancer progression. Type II High Grade Epithelial Ovarian Carcinomas (HG-EOC) represents the only clear exception to this observation. Here, we show that stathmin expression is necessary for the survival of HG-EOC cells carrying a p53 mutant (p53(MUT) ) gene. At molecular level, stathmin favours the binding and the phosphorylation of p53(MUT) by DNA-PKCS , eventually modulating p53(MUT) stability and transcriptional activity. Inhibition of stathmin or DNA-PKCS impaired p53(MUT) -dependent transcription of several M phase regulators, resulting in M phase failure and EOC cell death, both in vitro and in vivo. In primary human EOC a strong correlation exists between stathmin, DNA-PKCS , p53(MUT) overexpression and its transcriptional targets, further strengthening the relevance of the new pathway here described. Overall our data support the hypothesis that the expression of stathmin and p53 could be useful for the identification of high risk patients that will benefit from a therapy specifically acting on mitotic cancer cells.

  5. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

    Science.gov (United States)

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C

    2000-06-01

    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  6. Localizing potentially active post-transcriptional regulations in the Ewing's sarcoma gene regulatory network

    Directory of Open Access Journals (Sweden)

    Delyon Bernard

    2010-11-01

    Full Text Available Abstract Background A wide range of techniques is now available for analyzing regulatory networks. Nonetheless, most of these techniques fail to interpret large-scale transcriptional data at the post-translational level. Results We address the question of using large-scale transcriptomic observation of a system perturbation to analyze a regulatory network which contained several types of interactions - transcriptional and post-translational. Our method consisted of post-processing the outputs of an open-source tool named BioQuali - an automatic constraint-based analysis mimicking biologist's local reasoning on a large scale. The post-processing relied on differences in the behavior of the transcriptional and post-translational levels in the network. As a case study, we analyzed a network representation of the genes and proteins controlled by an oncogene in the context of Ewing's sarcoma. The analysis allowed us to pinpoint active interactions specific to this cancer. We also identified the parts of the network which were incomplete and should be submitted for further investigation. Conclusions The proposed approach is effective for the qualitative analysis of cancer networks. It allows the integrative use of experimental data of various types in order to identify the specific information that should be considered a priority in the initial - and possibly very large - experimental dataset. Iteratively, new dataset can be introduced into the analysis to improve the network representation and make it more specific.

  7. Viral retasking of hBre1/RNF20 to recruit hPaf1 for transcriptional activation.

    Directory of Open Access Journals (Sweden)

    Gregory J Fonseca

    Full Text Available Upon infection, human adenovirus (HAdV must activate the expression of its early genes to reprogram the cellular environment to support virus replication. This activation is orchestrated in large part by the first HAdV gene expressed during infection, early region 1A (E1A. E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. Previously, we identified hBre1/RNF20 as a target for E1A. The interaction between E1A and hBre1 antagonizes the innate antiviral response by blocking H2B monoubiquitination, a chromatin modification necessary for the interferon (IFN response. Here, we describe a second distinct role for the interaction of E1A with hBre1 in transcriptional activation of HAdV early genes. Furthermore, we show that E1A changes the function of hBre1 from a ubiquitin ligase involved in substrate selection to a scaffold which recruits hPaf1 as a means to stimulate transcription and transcription-coupled histone modifications. By using hBre1 to recruit hPaf1, E1A is able to optimally activate viral early transcription and begin the cycle of viral replication. The ability of E1A to target hBre1 to simultaneously repress cellular IFN dependent transcription while activating viral transcription, represents an elegant example of the incredible economy of action accomplished by a viral regulatory protein through a single protein interaction.

  8. Analysis of Polygenic Mutants Suggests a Role for Mediator in Regulating Transcriptional Activation Distance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Reavey, Caitlin T; Hickman, Mark J; Dobi, Krista C; Botstein, David; Winston, Fred

    2015-10-01

    Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.

  9. Transcriptional activation of hTERT in breast carcinomas by the Her2-ER81-related pathway.

    Science.gov (United States)

    Vageli, Dimitra; Ioannou, Maria G; Koukoulis, George K

    2009-01-01

    Her2 and ER81 (a member of ETS family) have been suggested to cause a synergistic increase in the transcriptional activation of hTERT. Our study aimed to offer further confirmation in clinical material. We determined the mRNA levels of Her2, ER81, and hTERT, by QRT-PCR, in 43 breast carcinomas. In the specimens showing hTERT transcriptional activation, Her2 and ER81 were increased in statistically significant tumor subgroups (61% and 79% correspondingly). The 86% of specimens with both Her2 and ER81 increased expression showed hTERT transcriptional activation. Synchronous transcriptional activation of hTERT, Her2, and ER81 elevated expression was noted in 42% of the samples. In conclusion, we agree with a previous study that Her2 overexpression may increase the hTERT transcriptional activation. Our data indicate that the mechanism may involve Her2-ER81 interaction(s) and that the activation of hTERT could be mainly mediated by transcriptional activation of ER81.

  10. PAX5 is the transcriptional activator of mucolipin-2 (MCOLN2) gene.

    Science.gov (United States)

    Valadez, Jessica A; Cuajungco, Math P

    2015-01-25

    Transient receptor potential mucolipin (TRPML) proteins belong to the TRP superfamily of non-selective cation channels. The TRPML1, -2, and -3 proteins are encoded by Mucolipin (MCOLN)-1, -2 and -3 genes, respectively. TRPML1 has been associated with mucolipidosis type IV (MLIV), while no disease phenotype has been linked with TRPML2 or -3 protein. The TRPML proteins share high sequence similarities, form hetero-tetramers, and serve in membrane trafficking, autophagy, and metal homeostasis. Previous studies suggest that TRPML2 serves a role in the immune system; however, the evidence is mostly indirect. We hypothesize that if TRPML2 is involved in immune function its expression would be likely regulated by an immune-associated transcription factor protein. Thus, we set out to identify the core promoter region and the transcription factor responsible for MCOLN2 gene expression. Using dual-luciferase assay and over-expression analyses, we reveal for the first time that B-cell lineage specific activator protein (BSAP), also known as paired box 5 (PAX5), controls MCOLN2 expression. Specifically, heterologous expression of PAX5 in HEK-293 cells significantly increased endogenous MCOLN2 transcript and TRPML2 protein levels, while RNA interference targeting endogenous PAX5 reduced its effect. Site-directed mutagenesis studies showed that the core promoter and PAX5 binding region to be between -79 and -60 base pairs upstream of the transcriptional start site. Thus, our findings add to a growing list of evidence for TRPML2's possible involvement in the immune system. The knowledge gained from this study could be used to further characterize the role of TRPML2 in B-cell development and function.

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

    Science.gov (United States)

    Wang, Fangfang; Okawa, Hiroko; Kamano, Yuya; Niibe, Kunimichi; Kayashima, Hiroki; Osathanon, Thanaphum; Pavasant, Prasit; Saeki, Makio; Yatani, Hirofumi; Egusa, Hiroshi

    2015-01-01

    Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation

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

    Directory of Open Access Journals (Sweden)

    Fangfang Wang

    Full Text Available Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR. Expression vectors that contained the Tet operator and amelogenin-coding (Amelx cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx. MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP, osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional

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

    Science.gov (United States)

    Wang, Fangfang; Okawa, Hiroko; Kamano, Yuya; Niibe, Kunimichi; Kayashima, Hiroki; Osathanon, Thanaphum; Pavasant, Prasit; Saeki, Makio; Yatani, Hirofumi; Egusa, Hiroshi

    2015-01-01

    Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation

  14. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene

    DEFF Research Database (Denmark)

    Soravia, E; Grebe, A; De Luca, P;

    1995-01-01

    The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we...... have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes...

  15. Promoter-proximal transcription factor binding is transcriptionally active when coupled with nucleosome repositioning in immediate vicinity

    Science.gov (United States)

    Yadav, Vinod Kumar; Thakur, Ram Krishna; Eckloff, Bruce; Baral, Aradhita; Singh, Ankita; Halder, Rashi; Kumar, Akinchan; Alam, Mohammad Parwez; Kundu, Tapas K.; Pandita, Raj; Pandita, Tej K.; Wieben, Eric D.; Chowdhury, Shantanu

    2014-01-01

    Previous studies have analyzed patterns of transcription, transcription factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy and do all TF binding and associated changes in nucleosome occupancy result in altered gene expression? With these in mind, following characterization of two states (before and after induction of a single TF of choice) we determined: (i) genomic binding sites of the TF, (ii) promoter nucleosome occupancy and (iii) transcriptome profiles. Results demonstrated that promoter-proximal TF binding influenced expression of the target gene when it was coupled to nucleosome repositioning at or close to its binding site in most cases. In contrast, only in few cases change in target gene expression was found when TF binding occurred without local nucleosome reorganization. PMID:25081206

  16. The Baltic Sea Virome: Diversity and Transcriptional Activity of DNA and RNA Viruses

    Science.gov (United States)

    McCrow, John P.; Ininbergs, Karolina; Dupont, Christopher L.; Badger, Jonathan H.; Hoffman, Jeffery M.; Ekman, Martin; Allen, Andrew E.; Bergman, Birgitta; Venter, J. Craig

    2017-01-01

    ABSTRACT Metagenomic and metatranscriptomic data were generated from size-fractionated samples from 11 sites within the Baltic Sea and adjacent marine waters of Kattegat and freshwater Lake Torneträsk in order to investigate the diversity, distribution, and transcriptional activity of virioplankton. Such a transect, spanning a salinity gradient from freshwater to the open sea, facilitated a broad genome-enabled investigation of natural as well as impacted aspects of Baltic Sea viral communities. Taxonomic signatures representative of phages within the widely distributed order Caudovirales were identified with enrichments in lesser-known families such as Podoviridae and Siphoviridae. The distribution of phage reported to infect diverse and ubiquitous heterotrophic bacteria (SAR11 clades) and cyanobacteria (Synechococcus sp.) displayed population-level shifts in diversity. Samples from higher-salinity conditions (>14 practical salinity units [PSU]) had increased abundances of viruses for picoeukaryotes, i.e., Ostreococcus. These data, combined with host diversity estimates, suggest viral modulation of diversity on the whole-community scale, as well as in specific prokaryotic and eukaryotic lineages. RNA libraries revealed single-stranded DNA (ssDNA) and RNA viral populations throughout the Baltic Sea, with ssDNA phage highly represented in Lake Torneträsk. Further, our data suggest relatively high transcriptional activity of fish viruses within diverse families known to have broad host ranges, such as Nodoviridae (RNA), Iridoviridae (DNA), and predicted zoonotic viruses that can cause ecological and economic damage as well as impact human health. IMPORTANCE Inferred virus-host relationships, community structures of ubiquitous ecologically relevant groups, and identification of transcriptionally active populations have been achieved with our Baltic Sea study. Further, these data, highlighting the transcriptional activity of viruses, represent one of the more

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

    Energy Technology Data Exchange (ETDEWEB)

    Iwahara, Naotoshi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Hisahara, Shin; Hayashi, Takashi [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Department of Neurology, Sapporo Medical University, Sapporo 060-8556 (Japan); Horio, Yoshiyuki, E-mail: horio@sapmed.ac.jp [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan)

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  18. Sequential Recruitment and Combinatorial Assembling of Multiprotein Complexes in Transcriptional Activation

    Science.gov (United States)

    Lemaire, Vincent; Lee, Chiu Fan; Lei, Jinzhi; Métivier, Raphaël; Glass, Leon

    2006-05-01

    In human cells, estrogenic signals induce cyclical association and dissociation of specific proteins with the DNA in order to activate transcription of estrogen-responsive genes. These oscillations can be modeled by assuming a large number of sequential reactions represented by linear kinetics with random kinetic rates. Application of the model to experimental data predicts robust binding sequences in which proteins associate with the DNA at several different phases of the oscillation. Our methods circumvent the need to derive detailed kinetic graphs, and are applicable to other oscillatory biological processes involving a large number of sequential steps.

  19. Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing.

    Science.gov (United States)

    Sun, Ning; Zhao, Huimin

    2013-07-01

    Transcription activator-like effector (TALE) nucleases (TALENs) have recently emerged as a revolutionary genome editing tool in many different organisms and cell types. The site-specific chromosomal double-strand breaks introduced by TALENs significantly increase the efficiency of genomic modification. The modular nature of the TALE central repeat domains enables researchers to tailor DNA recognition specificity with ease and target essentially any desired DNA sequence. Here, we comprehensively review the development of TALEN technology in terms of scaffold optimization, DNA recognition, and repeat array assembly. In addition, we provide some perspectives on the future development of this technology.

  20. Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs).

    Science.gov (United States)

    Taheri-Ghahfarokhi, Amir; Malaver-Ortega, Luis F; Sumer, Huseyin

    2015-01-01

    Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.

  1. The application of transcription activator-like effector nucleases for genome editing in C. elegans.

    Science.gov (United States)

    Yi, Peishan; Li, Wei; Ou, Guangshuo

    2014-08-01

    The nematode Caenorhabditis elegans has been a powerful model system for biomedical research in the past decades, however, the efficient genetic tools are still demanding for gene knockout, knock-in or conditional gene mutations. Transcription activator-like effector nucleases (TALENs) that comprise a sequence-specific DNA-binding domain fused to a FokI nuclease domain facilitate the targeted genome editing in various cell types or organisms. Here we summarize the recent progresses and protocols using TALENs in C. elegans that generate gene mutations and knock-ins in the germ line and the conditional gene knockout in somatic tissues.

  2. Germ cell nuclear factor directly represses the transcription of peroxisome proliferator-activated receptor delta gene

    Institute of Scientific and Technical Information of China (English)

    Chengqiang He; Naizheng Ding; Jie Kang

    2008-01-01

    Germ cell nuclear factor (GCNF) is a transcription factor that can repress gene transcription and plays an important role during spermatogenesis. Peroxisome proliferator-activated receptor delta (PPARδ) is a nuclear hormone receptor belonging to the steroid receptor superfamily.It can activate the expression of many genes,including those involved in lipid metabolism.In this report,we showed that GCNF specifically interacts with PPARδ promoter.Overexpression of GCNF in African green monkey SV40 transformed kidney fibroblast COS7 cells and mouse embryo fibroblast NIH 3T3 cells represses the activity of PPARδ promoter.The mutation of GCNF response element in PPARδ promoter relieves the repression in NIH 3T3 cells and mouse testis.Moreover,we showed that GCNF in nuclear extracts of mouse testis is able to bind to PPARδ promoter directly.We also found that GCNF and PPARδ mRNA were expressed with different patterns in mouse testis by in situ hybridization.These results suggested that GCNF might be a negative regulator of PPARδ gene expression through its direct interaction with PPARδ promoter in mouse testis.

  3. Structural Features and Transcriptional Activity of Chicken PPARs (α, β, and γ

    Directory of Open Access Journals (Sweden)

    Ichiro Takada

    2013-01-01

    Full Text Available While an understanding of lipid metabolism in chickens is critical for a further improvement of food production, there are few studies concerning differences in lipid metabolism mechanisms between chickens and other species at a molecular level. Chickens have three PPAR gene subtypes (α, β, and γ that function differently from those present in humans and mice. The chicken PPAR-gamma (cPPARγ gene is shorter than that in humans and lacks a γ2 isoform. Moreover, in serum-free media, cPPARγ shows high transcriptional activity without exogenous ligands. Luciferase reporter assays were used to examine the effect of sera on cPPAR transcriptional activities and showed that adult bovine serum and chicken serum highly activate cPPARα and β functions. Moreover, we found that bezafibrate induces the transactivation function of cPPARβ, but not human PPARδ (human PPARβ ortholog. This ligand selectivity relies on one amino acid residue (chicken: Val419, human: Met444. These results show the possibilities for unique functions of cPPARs on chicken-specific lipid glucose metabolism. As such, a better understanding of the molecular mechanisms of lipid metabolism in chickens could result in higher productivity for the poultry industry.

  4. Insulators target active genes to transcription factories and polycomb-repressed genes to polycomb bodies.

    Directory of Open Access Journals (Sweden)

    Hua-Bing Li

    2013-04-01

    Full Text Available Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs, mediate associations among Polycomb Group (PcG targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.

  5. Negative regulation of TLR-signaling pathways by activating transcription factor-3.

    Science.gov (United States)

    Whitmore, Mark M; Iparraguirre, Amaya; Kubelka, Lindsey; Weninger, Wolfgang; Hai, Tsonwin; Williams, Bryan R G

    2007-09-15

    Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.

  6. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants.

    Science.gov (United States)

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

  7. Dynamic transcription factor activity and networks during ErbB2 breast oncogenesis and targeted therapy.

    Science.gov (United States)

    Weiss, M S; Peñalver Bernabé, B; Shin, S; Asztalos, S; Dubbury, S J; Mui, M D; Bellis, A D; Bluver, D; Tonetti, D A; Saez-Rodriguez, J; Broadbelt, L J; Jeruss, J S; Shea, L D

    2014-12-01

    Tissue development and disease progression are multi-stage processes controlled by an evolving set of key regulatory factors, and identifying these factors necessitates a dynamic analysis spanning relevant time scales. Current omics approaches depend on incomplete biological databases to identify critical cellular processes. Herein, we present TRACER (TRanscriptional Activity CEll aRrays), which was employed to quantify the dynamic activity of numerous transcription factor (TFs) simultaneously in 3D and networks for TRACER (NTRACER), a computational algorithm that allows for cellular rewiring to establish dynamic regulatory networks based on activity of TF reporter constructs. We identified major hubs at various stages of culture associated with normal and abnormal tissue growth (i.e., ELK-1 and E2F1, respectively) and the mechanism of action for a targeted therapeutic, lapatinib, through GATA-1, which were confirmed in human ErbB2 positive breast cancer patients and human ErbB2 positive breast cancer cell lines that were either sensitive or resistant to lapatinib.

  8. Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotrophin-mediated gene expression.

    Science.gov (United States)

    Groth, Rachel D; Mermelstein, Paul G

    2003-09-03

    A member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) regulates neuronal survival and differentiation during development. Within the adult brain, BDNF is also important in neuronal adaptive processes, such as the activity-dependent plasticity that underlies learning and memory. These long-term changes in synaptic strength are mediated through alterations in gene expression. However, many of the mechanisms by which BDNF is linked to transcriptional and translational regulation remain unknown. Recently, the transcription factor NFATc4 (nuclear factor of activated T-cells isoform 4) was discovered in neurons, where it is believed to play an important role in long-term changes in neuronal function. Interestingly, NFATc4 is particularly sensitive to the second messenger systems activated by BDNF. Thus, we hypothesized that NFAT-dependent transcription may be an important mediator of BDNF-induced plasticity. In cultured rat CA3-CA1 hippocampal neurons, BDNF activated NFAT-dependent transcription via TrkB receptors. Inhibition of calcineurin blocked BDNF-induced nuclear translocation of NFATc4, thus preventing transcription. Further, phospholipase C was a critical signaling intermediate between BDNF activation of TrkB and the initiation of NFAT-dependent transcription. Both inositol 1,4,5-triphosphate (IP3)-mediated release of calcium from intracellular stores and activation of protein kinase C were required for BDNF-induced NFAT-dependent transcription. Finally, increased expression of IP3 receptor 1 and BDNF after neuronal exposure to BDNF was linked to NFAT-dependent transcription. These results suggest that NFATc4 plays a crucial role in neurotrophin-mediated synaptic plasticity.

  9. Exercise induces transient transcriptional activation of the PGC-1a gene in human skeletal muscle

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P. Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1a (PGC-1a) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell...... culture and rodent skeletal muscle. To determine whether PGC-1a transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two......-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl...

  10. STAT5 transcriptional activity is impaired by LIF in a mammary epithelial cell line.

    Science.gov (United States)

    Granillo, Agustina Rodriguez; Boffi, Juan Carlos; Barañao, Lino; Kordon, Edith; Pecci, Adali; Guberman, Alejandra

    2007-05-11

    Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation. We have also found that STAT5 activity is negatively modulated by this cytokine, both on a gene whose expression is induced, as well as on a promoter repressed by STAT5. Besides, our results show that lactogenic hormones increase LIF effect on gene induction without modifying STAT3 phosphorylation state. Our findings strongly suggest that there is crosstalk between STAT5 and STAT3 pathways that could modulate their ability to regulate gene expression.

  11. Signal transducer and activator of transcription 3 regulation by novel binding partners

    Institute of Scientific and Technical Information of China (English)

    Tadashi; Matsuda; Ryuta; Muromoto; Yuichi; Sekine; Sumihito; Togi; Yuichi; Kitai; Shigeyuki; Kon; Kenji; Oritani

    2015-01-01

    Signal transducers and activators of transcription(STATs) mediate essential signals for various biological processes,including immune responses,hematopoiesis,and neurogenesis. STAT3,for example,is involved in the pathogenesis of various human diseases,including cancers,autoimmune and inflammatory disorders. STAT3 activation is therefore tightly regulated at multiple levels to prevent these pathological conditions. A number of proteins have been reported to associate with STAT3 and regulate its activity. These STAT3-interacting proteins function to modulate STAT3-mediated signaling at various steps and mediate the crosstalk of STAT3 with other cellular signaling pathways. This article reviews the roles of novel STAT3 binding partners such as DAXX,zipperinteracting protein kinase,Krüppel-associated box-associated protein 1,Y14,PDZ and LIM domain 2 and signal transducing adaptor protein-2,in the regulation of STAT3-mediated signaling.

  12. PEA3 activates VEGF transcription in T47D and SKBR3 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Dong Hua; Bobin Chen; Mei Bai; Hao Yu; Xiaohong Wu; Wei Jin

    2009-01-01

    Vascular endothelial growth factor(VEGF)is a potent stimulator of angiogenesis and a prognostic factor for many tumors,including those of endocrine-responsive tissues such as the breast and uterus.In this study,we found that overexpression of PEA3 could increase VEGF mRNA levels and VEGF promoter activity in human T47D and SKBR3 breast cancer cells.Chromatin immunoprecipitation assay demonstrated that PEA3 could bind to the VEGF promoter in the cells transfected with PEA3 expression vector.PEA3 small interfering RNA attenuated VEGF promoter activity and the binding of PEA3 to the VEGF promoter in T47D and SKBR3 cells.These results indicated that PEA3 could activate VEGF promoter transcription.

  13. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong, E-mail: yelihong@nankai.edu.cn [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2013-05-03

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

  14. Mdm2 RING mutation enhances p53 transcriptional activity and p53-p300 interaction.

    Directory of Open Access Journals (Sweden)

    Hilary V Clegg

    Full Text Available The p53 transcription factor and tumor suppressor is regulated primarily by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 to target it for proteasomal degradation. Aside from its ubiquitin ligase function, Mdm2 has been believed to be capable of suppressing p53's transcriptional activity by binding with and masking the transactivation domain of p53. The ability of Mdm2 to restrain p53 activity by binding alone, without ubiquitination, was challenged by a 2007 study using a knockin mouse harboring a single cysteine-to-alanine point mutation (C462A in Mdm2's RING domain. Mouse embryonic fibroblasts with this mutation, which abrogates Mdm2's E3 ubiquitin ligase activity without affecting its ability to bind with p53, were unable to suppress p53 activity. In this study, we utilized the Mdm2(C462A mouse model to characterize in further detail the role of Mdm2's RING domain in the control of p53. Here, we show in vivo that the Mdm2(C462A protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2(C462A actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2(C462A facilitates the interaction between p53 and the acetyltransferase CBP/p300, and it fails to heterodimerize with its homolog and sister regulator of p53, Mdmx, suggesting that a fully intact RING domain is required for Mdm2's inhibition of the p300-p53 interaction and for its interaction with Mdmx. These findings help us to better understand the complex regulation of the Mdm2-p53 pathway and have important implications for chemotherapeutic agents targeting Mdm2, as they suggest that inhibition of Mdm2's E3 ubiquitin ligase activity may be sufficient for increasing p53 activity in vivo, without the need to block Mdm2-p53 binding.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1...... cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced......-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate...

  16. Statistical reconstruction of transcription factor activity using Michaelis-Menten kinetics.

    Science.gov (United States)

    Khanin, R; Vinciotti, V; Mersinias, V; Smith, C P; Wit, E

    2007-09-01

    The basic building block of a gene regulatory network consists of a gene encoding a transcription factor (TF) and the gene(s) it regulates. Considerable efforts have been directed recently at devising experiments and algorithms to determine TFs and their corresponding target genes using gene expression and other types of data. The underlying problem is that the expression of a gene coding for the TF provides only limited information about the activity of the TF, which can also be controlled posttranscriptionally. In the absence of a reliable technology to routinely measure the activity of regulators, it is of great importance to understand whether this activity can be inferred from gene expression data. We here develop a statistical framework to reconstruct the activity of a TF from gene expression data of the target genes in its regulatory module. The novelty of our approach is that we embed the deterministic Michaelis-Menten model of gene regulation in this statistical framework. The kinetic parameters of the gene regulation model are inferred together with the profile of the TF regulator. We also obtain a goodness-of-fit test to verify the fit of the model. The model is applied to a time series involving the Streptomyces coelicolor bacterium. We focus on the transcriptional activator cdaR, which is partly responsible for the production of a particular type of antibiotic. The aim is to reconstruct the activity profile of this regulator. Our approach can be extended to include more complex regulatory relationships, such as multiple regulatory factors, competition, and cooperativity.

  17. A role for RNA post-transcriptional regulation in satellite cell activation

    Directory of Open Access Journals (Sweden)

    Farina Nicholas H

    2012-10-01

    Full Text Available Abstract Background Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. Methods Satellite cells isolated by FACS from uninjured skeletal muscle and 12 h post-muscle injury from wild type and Syndecan-4 null mice were probed using Affymetrix 430v2 gene chips and analyzed by Spotfiretm and Ingenuity Pathway analysis to identify gene expression changes and networks associated with satellite cell activation, respectively. Additional analyses of target genes identify miRNAs exhibiting dynamic changes in expression during satellite cell activation. The function of the miRNAs was assessed using miRIDIAN hairpin inhibitors. Results An unbiased gene expression screen identified over 4,000 genes differentially expressed in satellite cells in vivo within 12 h following muscle damage and more than 50% of these decrease dramatically. RNA binding proteins and genes involved in post-transcriptional regulation were significantly over-represented whereas splicing factors were preferentially downregulated and mRNA stability genes preferentially upregulated. Furthermore, six computationally identified miRNAs demonstrated novel expression through muscle regeneration and in satellite cells. Three of the six miRNAs were found to regulate satellite cell fate. Conclusions The quiescent satellite cell is actively maintained in a state poised to activate in response to external signals. Satellite cell activation appears to be regulated by post-transcriptional gene regulation.

  18. A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta.

    Science.gov (United States)

    Zhang, Lei; Davies, Laura J; Elling, Axel A

    2015-01-01

    Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-β-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity.

  19. Menin and RNF20 recruitment is associated with dynamic histone modifications that regulate signal transducer and activator of transcription 1 (STAT1-activated transcription of the interferon regulatory factor 1 gene (IRF1

    Directory of Open Access Journals (Sweden)

    Buro Lauren J

    2010-09-01

    Full Text Available Abstract Background Signal transducer and activator of transcription (STAT activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1. Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFNγ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

  20. Transcriptional activation of glutathione pathways and role of glucose homeostasis during copper imbalance.

    Science.gov (United States)

    Quiroz, Natalia; Rivas, Nicole; del Pozo, Talía; Burkhead, Jason; Suazo, Miriam; González, Mauricio; Latorre, Mauricio

    2015-04-01

    Copper is an essential micronutrient for organism health. Dietary changes or pathologies linked to this metal induce changes in intracellular glutathione concentrations. Here, we studied the transcriptional activation of glutathione pathways in Jurkat cell lines, analyzing the effect of change in glucose homeostasis during a physiological and supra-physiological copper exposure. An immortalized line of human T lymphocyte cell line (Jurkat) was exposed to different copper and glucose conditions to mimic concentrations present in human blood. We applied treatments for 6 (acute) and 24 h (sustained) to 2 µM (physiological) or 20 µM (supra-physiological, Wilson disease scenario) of CuSO4 in combination with 25 mg/dL (hypoglycemia), 100 mg/dL (normal) and 200 mg/dL (hyperglycemia, diabetes scenario) of glucose. The results indicate that a physiological concentration of copper exposure does not induce transcriptional changes in the glutathione synthesis pathway after 6 or 24 h. The G6PDH gene (regeneration pathway), however, is induced during a supra-physiological copper condition. This data was correlated with the viability assays, where fluctuation in both glucose conditions (hypo and hyperglycemia scenario) affected Jurkat proliferation when 20 µM of CuSO4 was added to the culture media. Under a copper overload condition, the transcription of a component of glutathione regeneration pathway (G6PDH gene) is activated in cells chronically exposed to a hyperglycemia scenario, indicating that fluctuations in glucose concentration impact the resistance against the metal. Our findings illustrate the importance of glucose homeostasis during copper excess.

  1. Pronounced cohabitation of active immunoglobulin genes from three different chromosomes in transcription factories during maximal antibody synthesis.

    Science.gov (United States)

    Park, Sung-Kyun; Xiang, Yougui; Feng, Xin; Garrard, William T

    2014-06-01

    To understand the relationships between nuclear organization and gene expression in a model system, we employed three-dimensional imaging and chromatin immunoprecipitation (ChIP)-chromosome conformation capture (3C) techniques to investigate the topographies of the immunoglobulin (Ig) genes and transcripts during B-cell development. Remarkably, in plasma cells, when antibody synthesis peaks, active Ig genes residing on three different chromosomes exhibit pronounced colocalizations in transcription factories, often near the nuclear periphery, and display trans-chromosomal enhancer interactions, and their transcripts frequently share interchromatin trafficking channels. Conceptually, these features of nuclear organization maximize coordinated transcriptional and transcript trafficking control for potentiating the optimal cytoplasmic assembly of the resulting translation products into protein multimers.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Xu-Dong [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Meng, Qing-Hui [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Xu, Jia-Ying; Jiao, Yang [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Ge, Chun-Min [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Jacob, Asha; Wang, Ping [North Shore University Hospital-Long Island Jewish Medical Center and The Feinstein Institute for Medical Research, Manhasset, NY 11030 (United States); Rosen, Eliot M [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Fan, Saijun, E-mail: sjfan@suda.edu.cn [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China)

    2011-01-28

    Research highlights: {yields} BTG2 associates with AR, androgen causes an increase of the interaction. {yields} BTG2 as a co-repressor inhibits the AR-mediated transcription activity. {yields} BTG2 inhibits the transcription activity and expression of PSA. {yields} An intact {sup 92}LxxLL{sup 96} motif is essential and necessary for these activities of BTG2, while the {sup 20}LxxLL{sup 24} motif is not required. {yields} Ectopic expression of BTG2 reduces proliferation of prostate cancer cells. -- Abstract: The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ({sup 20}LxxLL{sup 24} and {sup 92}LxxLL{sup 96}), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5{alpha}-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant {sup 20}LxxLL{sup 24} motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant {sup 92}LxxLL{sup 96} motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact {sup 92}LxxLL{sup 96} motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

  3. Measuring riboswitch activity in vitro and in artificial cells with purified transcription-translation machinery.

    Science.gov (United States)

    Martini, Laura; Mansy, Sheref S

    2014-01-01

    We present a simple method to measure the real-time activity of riboswitches with purified components in vitro and inside of artificial cells. Typically, riboswitch activity is measured in vivo by exploiting β-galactosidase encoding constructs with a putative riboswitch sequence in the untranslated region. Additional in vitro characterization often makes use of in-line probing to explore conformational changes induced by ligand binding to the mRNA or analyses of transcript lengths in the presence and absence of ligand. However, riboswitches ultimately control protein levels and often times require accessory factors. Therefore, an in vitro system capable of monitoring protein production with fully defined components that can be supplemented with accessory factors would greatly aid riboswitch studies. Herein we present a system that is amenable to such analyses. Further, since the described system can be easily reconstituted within compartments to build artificial, cellular mimics with sensing capability, protocols are provided for building sense-response systems within water-in-oil emulsion compartments and lipid vesicles. Only standard laboratory equipment and commercially available material are exploited for the described assays, including DNA, purified transcription-translation machinery, i.e., the PURE system, and a spectrofluorometer.

  4. Transcription Activity of Ectogenic Human Carcinoembryonic Antigen Promoter in Lung Adenocarcinoma Cells A549

    Institute of Scientific and Technical Information of China (English)

    XIONG Weining; FANG Huijuan; XU Yongjian; XIONG Shendao; CAO Yong; SONG Qingfeng; ZENG Daxiong; ZHANG Huilan

    2006-01-01

    The transcription activity of ectogenic human carcinoembryonic antigen (CEA) promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein (GFP) driven by CEA promoter and human cytomegalovirus (CMV) promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed (4.08±0.63) % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [(43.27±3.54) %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.

  5. ACETYLATION INCREASES EWS-FLI1 DNA BINDING AND TRANSCRIPTIONAL ACTIVITY

    Directory of Open Access Journals (Sweden)

    Silke eSchlottmann

    2012-09-01

    Full Text Available Ewing Sarcoma (ES is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant posttranslational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors, and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with histone deacetylase inhibitors (HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in COS7 cells. However, our data that evaluates the acetylation of ful-length EWS-FLI1 remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  6. SUMOylation modulates the transcriptional activity of androgen receptor in a target gene and pathway selective manner.

    Science.gov (United States)

    Sutinen, Päivi; Malinen, Marjo; Heikkinen, Sami; Palvimo, Jorma J

    2014-07-01

    Androgen receptor (AR) plays an important regulatory role in prostate cancer. AR's transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates AR's interaction with the chromatin and the receptor's target gene selection.

  7. GFP tracking transcriptional activity endogenous p53: vector construction and application in genotoxicity detection

    Institute of Scientific and Technical Information of China (English)

    ZENG Wei-sen; LUO Chen; XIE Wei-bing; CHEN Han-yuan

    2001-01-01

    To establish a sensitive.and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H202 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H202, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP, hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.

  8. Conserved hypothetical BB0462 protein enhances the transcription activity of oppAV promoter

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Borrelia burgdorferi BB0462 ORF encodes an unknown functional protein with 110 amino acids.A BLAST search in protein databases and the secondary structure being predicted by the program JUFO showed that the conserved hypothetical BB0462 protein was similar to the members of the YbaB protein family in both amino acid composition and protein structure.The co-transformation of BB0462 ORF and oppA upstream regulation DNA into E.coli host cells and β-galactosidase activity assay demonstrated that the BB0462 protein enhanced the transcriptional activity of the oppAV promoter,but does not affect those of oppAⅠ,Ⅱ,Ⅲ and Ⅳ promoters.Analysis of DNA retardation and competitive repression also confirmed that the BB0462 protein bound to the 409 bp upstream regulation DNA fragment close to the initiation codon of the oppAV gene.All data in our study suggested that the BB0462 protein was involved in the transcriptional regulation of the oppAV gene

  9. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    Science.gov (United States)

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

  10. Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers.

    Science.gov (United States)

    Bultmann, Sebastian; Morbitzer, Robert; Schmidt, Christine S; Thanisch, Katharina; Spada, Fabio; Elsaesser, Janett; Lahaye, Thomas; Leonhardt, Heinrich

    2012-07-01

    Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2'-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes.

  11. ATPase site architecture is required for self-assembly and remodeling activity of a hexameric AAA+ transcriptional activator.

    Science.gov (United States)

    Joly, Nicolas; Zhang, Nan; Buck, Martin

    2012-08-10

    AAA+ proteins (ATPases associated with various cellular activities) are oligomeric ATPases that use ATP hydrolysis to remodel their substrates. By similarity with GTPases, a dynamic organization of the nucleotide-binding pockets between ATPase protomers is proposed to regulate functionality. Using the transcription activator PspF as an AAA+ model, we investigated contributions of conserved residues for roles in ATP hydrolysis and intersubunit communication. We determined the R-finger residue and revealed that it resides in a conserved "R-hand" motif (R(x)D(xxx)R) needed for its "trans-acting" activity. Further, a divergent Walker A glutamic acid residue acts synergistically with a tyrosine residue to function in ADP-dependent subunit-subunit coordination, forming the "ADP-switch" motif. Another glutamic acid controls hexamer formation in the presence of nucleotides. Together, these results lead to a "residue-nucleotide" interaction map upon which to base AAA+ core regulation.

  12. Identification and Characterization of Reverse Transcriptase Domain of Transcriptionally Active Retrotransposons in Wheat Genomes

    Institute of Scientific and Technical Information of China (English)

    Yi-Miao TANG; You-Zhi MA; Lian-Cheng LI; Xing-Guo YE

    2005-01-01

    To clarify activation characterization of wheat (Triticum aestivum L.) retrotransposons, transcriptionally active Ty1-copia retrotransposons were found in wheat by using RT-PCR to amplify the RT domain. Sequence analysis of random RT-PCR clones reveals that Ty1-copia retrotransposons are highly heterogeneous and can be divided into at least four groups, which are tentatively named TaRT-1 to TaRT-4.Dot blot hybridization indicates that TaRT- 1 exists in the wheat genome as multiple copies (at 30 000 copies/a hexaploid genome (ABD)). Northern blot hybridization showed that TaRT-1 is only expressed at a low level under normal conditions in seedlings, but at a high level when induced by powdery mildew fungus, jasmonic acid (JA) and salicylic acid (SA). These results suggest that the TaRT-1 expression is highly sensitive to biotic and abiotic stresses.

  13. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases.

    Science.gov (United States)

    Zhao, Xinxia; Ni, Wei; Chen, Chuangfu; Sai, Wujiafu; Qiao, Jun; Sheng, Jingliang; Zhang, Hui; Li, Guozhong; Wang, Dawei; Hu, Shengwei

    2016-03-01

    Myostatin (MSTN) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs) in tandem with single-stranded DNA oligonucleotides (ssODNs). We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  14. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

    Science.gov (United States)

    Dupuy, Bruno; Mani, Nagraj; Katayama, Seiichi; Sonenshein, Abraham L

    2005-02-01

    Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

  15. Hes-1, a known transcriptional repressor, acts as a transcriptional activator for the human acid alpha-glucosidase gene in human fibroblast cells.

    Science.gov (United States)

    Yan, Bo; Raben, Nina; Plotz, Paul H

    2002-03-01

    Hes-1, the mammalian homologue 1 of Drosophila hairy and Enhancer of split proteins, belongs to a family of basic helix-loop-helix proteins that are essential to neurogenesis, myogenesis, hematopoiesis, and sex determination. Hes-1 is a transcriptional repressor for a number of known genes including the human acid alpha-glucosidase (GAA) gene as we have previously shown in Hep G2 cells. The human GAA gene encodes the enzyme for glycogen breakdown in lysosomes, deficiency of which results in Glycogen Storage Disease type II (Pompe syndrome). Using constructs containing the DNA element that demonstrates repressive activity in Hep G2 cells and conditions in which the same transcription factors, Hes-1 and YY1, bind, we have shown that this element functions as an enhancer in human fibroblasts. Site-directed mutagenesis and overexpression of Hes-1 showed that Hes-1 functions as a transcriptional activator. The dual function of Hes-1 we have found is likely to contribute to the subtle tissue-specific control of this housekeeping gene.

  16. A cytoplasmic negative regulator isoform of ATF7 impairs ATF7 and ATF2 phosphorylation and transcriptional activity.

    Science.gov (United States)

    Diring, Jessica; Camuzeaux, Barbara; Donzeau, Mariel; Vigneron, Marc; Rosa-Calatrava, Manuel; Kedinger, Claude; Chatton, Bruno

    2011-01-01

    Alternative splicing and post-translational modifications are processes that give rise to the complexity of the proteome. The nuclear ATF7 and ATF2 (activating transcription factor) are structurally homologous leucine zipper transcription factors encoded by distinct genes. Stress and growth factors activate ATF2 and ATF7 mainly via sequential phosphorylation of two conserved threonine residues in their activation domain. Distinct protein kinases, among which mitogen-activated protein kinases (MAPK), phosphorylate ATF2 and ATF7 first on Thr71/Thr53 and next on Thr69/Thr51 residues respectively, resulting in transcriptional activation. Here, we identify and characterize a cytoplasmic alternatively spliced isoform of ATF7. This variant, named ATF7-4, inhibits both ATF2 and ATF7 transcriptional activities by impairing the first phosphorylation event on Thr71/Thr53 residues. ATF7-4 indeed sequesters the Thr53-phosphorylating kinase in the cytoplasm. Upon stimulus-induced phosphorylation, ATF7-4 is poly-ubiquitinated and degraded, enabling the release of the kinase and ATF7/ATF2 activation. Our data therefore conclusively establish that ATF7-4 is an important cytoplasmic negative regulator of ATF7 and ATF2 transcription factors.

  17. A cytoplasmic negative regulator isoform of ATF7 impairs ATF7 and ATF2 phosphorylation and transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jessica Diring

    Full Text Available Alternative splicing and post-translational modifications are processes that give rise to the complexity of the proteome. The nuclear ATF7 and ATF2 (activating transcription factor are structurally homologous leucine zipper transcription factors encoded by distinct genes. Stress and growth factors activate ATF2 and ATF7 mainly via sequential phosphorylation of two conserved threonine residues in their activation domain. Distinct protein kinases, among which mitogen-activated protein kinases (MAPK, phosphorylate ATF2 and ATF7 first on Thr71/Thr53 and next on Thr69/Thr51 residues respectively, resulting in transcriptional activation. Here, we identify and characterize a cytoplasmic alternatively spliced isoform of ATF7. This variant, named ATF7-4, inhibits both ATF2 and ATF7 transcriptional activities by impairing the first phosphorylation event on Thr71/Thr53 residues. ATF7-4 indeed sequesters the Thr53-phosphorylating kinase in the cytoplasm. Upon stimulus-induced phosphorylation, ATF7-4 is poly-ubiquitinated and degraded, enabling the release of the kinase and ATF7/ATF2 activation. Our data therefore conclusively establish that ATF7-4 is an important cytoplasmic negative regulator of ATF7 and ATF2 transcription factors.

  18. Parental origin of chromosomes influences crossover activity within the Kcnq1 transcriptionally imprinted domain of Mus musculus

    Directory of Open Access Journals (Sweden)

    Petkov Petko M

    2009-05-01

    Full Text Available Abstract Background Among the three functions of DNA, mammalian replication and transcription can be subject to epigenetic imprinting specified by the parental origin of chromosomes, and although there is suggestive indication that this is also true for meiotic recombination, no definitive evidence has yet been reported. Results We have now obtained such evidence on mouse chromosome 7 by assaying meiotic recombination as it occurs in reciprocal F1 mice. A 166 kb region near the Kcnq1 transcriptionally imprinted domain showed significantly higher recombination activity in the CAST×B6 parental direction (p Slc22a18 showed a definitive parent of origin effect on recombination frequency (p Kcnq1 and neighboring H19-Igf2 imprinted domains with their human counterparts, we found that elevated recombination activity in these domains is a consequence of their chromosomal position relative to the telomere and not an intrinsic characteristic of transcriptionally imprinted domains as has been previously suggested. Conclusion Similar to replication and transcription, we demonstrate that meiotic recombination can be subjected to epigenetic imprinting and hotspot activity can be influenced by the parental origin of chromosomes. Furthermore, transcriptionally imprinted regions exhibiting elevated recombination activity are likely a consequence of their chromosomal location rather than their transcriptional characteristic.

  19. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    Science.gov (United States)

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

  20. Glucocorticoid Receptor Transcriptional Activation via the BRG1-Dependent Recruitment of TOP2β and Ku70/86.

    Science.gov (United States)

    Trotter, Kevin W; King, Heather A; Archer, Trevor K

    2015-08-01

    BRG1, the central ATPase of the human SWI/SNF complex, is critical for biological functions, including nuclear receptor (NR)-regulated transcription. Analysis of BRG1 mutants demonstrated that functional motifs outside the ATPase domain are important for transcriptional activity. In the course of experiments examining protein interactions mediated through these domains, Ku70 (XRCC6) was found to associate with a BRG1 fragment encompassing the conserved helicase-SANT-associated (HSA) and BRK domains of BRG1. Subsequent transcriptional activation assays and chromatin immunoprecipitation studies showed that Ku70/86 and components of the topoisomerase IIβ (TOP2β)/poly(ADP ribose) polymerase 1 (PARP1) complex are necessary for NR-mediated SWI/SNF-dependent transcriptional activation from endogenous promoters. In addition to establishing Ku-BRG1 binding and TOP2β/PARP1 recruitment by nuclear receptor transactivation, we demonstrate that the transient appearance of glucocorticoid receptor (GR)/BRG1-dependent, TOP2β-mediated double-strand DNA breaks is required for efficient GR-stimulated transcription. Taken together, these results suggest that a direct interaction between Ku70/86 and BRG1 brings together SWI/SNF remodeling capabilities and TOP2β activity to enhance the transcriptional response to hormone stimulation.

  1. Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases.

    Science.gov (United States)

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-03-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44-100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1(Δ7/Δ7) fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.

  2. Loss of signal transducer and activator of transcription 3 (STAT3) signaling during elevated activity causes vulnerability in hippocampal neurons.

    Science.gov (United States)

    Murase, Sachiko; Kim, Eunyoung; Lin, Lin; Hoffman, Dax A; McKay, Ronald D

    2012-10-31

    Chronically altered levels of network activity lead to changes in the morphology and functions of neurons. However, little is known of how changes in neuronal activity alter the intracellular signaling pathways mediating neuronal survival. Here, we use primary cultures of rat hippocampal neurons to show that elevated neuronal activity impairs phosphorylation of the serine/threonine kinase, Erk1/2, and the activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylation of serine 727. Chronically stimulated neurons go through apoptosis when they fail to activate another serine/threonine kinase, Akt. Gain- and loss-of-function experiments show that STAT3 plays the key role directly downstream from Erk1/2 as the alternative survival pathway. Elevated neuronal activity resulted in increased expression of a tumor suppressor, p53, and its target gene, Bax. These changes are observed in Kv4.2 knock-out mouse hippocampal neurons, which are also sensitive to the blockade of TrkB signaling, confirming that the alteration occurs in vivo. Thus, this study provides new insight into a mechanism by which chronic elevation of activity may cause neurodegeneration.

  3. HBXIP up-regulates ACSL1 through activating transcriptional factor Sp1 in breast cancer.

    Science.gov (United States)

    Wang, Yue; Cai, Xiaoli; Zhang, Shuqin; Cui, Ming; Liu, Fabao; Sun, Baodi; Zhang, Weiying; Zhang, Xiaodong; Ye, Lihong

    2017-03-11

    The oncoprotein hepatitis B X-interacting protein (HBXIP) results in the dysregulation of lipid metabolism to enhance the development of breast cancer. Acyl-CoA synthetase long-chain family member 1 (ACSL1) is required for thioesterification of long-chain fatty acids into their acyl-CoA derivatives. In this study, we present a hypothesis that HBXIP might be involved in the regulation of ACSL1 in breast cancer. Interestingly, we found that the overexpression of HBXIP was able to up-regulate ACSL1 at the levels of mRNA and protein in a dose-dependent manner in breast cancer cells. Conversely, silencing of HBXIP led to the opposite results. Mechanistically, HBXIP as a coactivator interacted with transcriptional factor Sp1 through binding to the promoter of ACSL1 by ChIP assays analysis, leading to the transcription of ACSL1 in breast cancer cells. Immunohistochemistry staining revealed that the positive rate of ACSL1 was 71.4% (35/49) in clinical breast cancer tissues, HBXIP 79.6% (39/49), in which the positive rate of ACSL1 was 76.9% (30/39) in the HBXIP-positive specimens. But, few positive rate of ACSL1 10% (1/10) was observed in normal breast tissues. The mRNA levels of ACSL1 were significantly higher in clinical breast cancer tissues than those in their corresponding peritumor tissues. The mRNA levels of ACSL1 were positively associated with those of HBXIP in clinical breast cancer tissues. Thus, we conclude that the oncoprotein HBXIP is able to up-regulate ACSL1 through activating the transcriptional factor Sp1 in breast cancer.

  4. Release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein (snRNP) activates hexamethylene bisacetamide-inducible protein (HEXIM1) transcription.

    Science.gov (United States)

    Liu, Pingyang; Xiang, Yanhui; Fujinaga, Koh; Bartholomeeusen, Koen; Nilson, Kyle A; Price, David H; Peterlin, B Matija

    2014-04-04

    By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.

  5. Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.

    Science.gov (United States)

    O'Shea, Charlotte; Kryger, Mikael; Stender, Emil G P; Kragelund, Birthe B; Willemoës, Martin; Skriver, Karen

    2015-01-15

    Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013.

  6. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling

    OpenAIRE

    Lee, Elaine Choung-Hee; Strange, Kevin

    2012-01-01

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is ...

  7. Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases

    OpenAIRE

    Bloom, Kristie; Ely, Abdullah; Mussolino, Claudio; Cathomen, Toni; Arbuthnot, Patrick

    2013-01-01

    Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-...

  8. Dynamic, large-scale profiling of transcription factor activity from live cells in 3D culture.

    Directory of Open Access Journals (Sweden)

    Michael S Weiss

    Full Text Available BACKGROUND: Extracellular activation of signal transduction pathways and their downstream target transcription factors (TFs are critical regulators of cellular processes and tissue development. The intracellular signaling network is complex, and techniques that quantify the activities of numerous pathways and connect their activities to the resulting phenotype would identify the signals and mechanisms regulating tissue development. The ability to investigate tissue development should capture the dynamic pathway activity and requires an environment that supports cellular organization into structures that mimic in vivo phenotypes. Taken together, our objective was to develop cellular arrays for dynamic, large-scale quantification of TF activity as cells organized into spherical structures within 3D culture. METHODOLOGY/PRINCIPAL FINDINGS: TF-specific and normalization reporter constructs were delivered in parallel to a cellular array containing a well-established breast cancer cell line cultured in Matrigel. Bioluminescence imaging provided a rapid, non-invasive, and sensitive method to quantify luciferase levels, and was applied repeatedly on each sample to monitor dynamic activity. Arrays measuring 28 TFs identified up to 19 active, with 13 factors changing significantly over time. Stimulation of cells with β-estradiol or activin A resulted in differential TF activity profiles evolving from initial stimulation of the ligand. Many TFs changed as expected based on previous reports, yet arrays were able to replicate these results in a single experiment. Additionally, arrays identified TFs that had not previously been linked with activin A. CONCLUSIONS/SIGNIFICANCE: This system provides a method for large-scale, non-invasive, and dynamic quantification of signaling pathway activity as cells organize into structures. The arrays may find utility for investigating mechanisms regulating normal and abnormal tissue growth, biomaterial design, or as a

  9. Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo.

    Science.gov (United States)

    Nudelman, Aaron S; DiRocco, Derek P; Lambert, Talley J; Garelick, Michael G; Le, Josh; Nathanson, Neil M; Storm, Daniel R

    2010-04-01

    Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h of stimulation. Expression levels of primary and mature-miR-132 increased significantly between postnatal Days 10 and 24. We conclude that miR-132 is an activity-dependent microRNA in vivo, and may contribute to the long-lasting proteomic changes required for experience-dependent neuronal plasticity.

  10. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.

    Science.gov (United States)

    Zhong, Bo; Yang, Yan; Li, Shu; Wang, Yan-Yi; Li, Ying; Diao, Feici; Lei, Caoqi; He, Xiao; Zhang, Lu; Tien, Po; Shu, Hong-Bing

    2008-10-17

    Viral infection triggers activation of transcription factors such as NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. Here, we identified MITA as a critical mediator of virus-triggered type I IFN signaling by expression cloning. Overexpression of MITA activated IRF3, whereas knockdown of MITA inhibited virus-triggered activation of IRF3, expression of type I IFNs, and cellular antiviral response. MITA was found to localize to the outer membrane of mitochondria and to be associated with VISA, a mitochondrial protein that acts as an adaptor in virus-triggered signaling. MITA also interacted with IRF3 and recruited the kinase TBK1 to the VISA-associated complex. MITA was phosphorylated by TBK1, which is required for MITA-mediated activation of IRF3. Our results suggest that MITA is a critical mediator of virus-triggered IRF3 activation and IFN expression and further demonstrate the importance of certain mitochondrial proteins in innate antiviral immunity.

  11. Signal transducer and activator of transcription 5B (STAT5B) modulates adipocyte differentiation via MOF.

    Science.gov (United States)

    Gao, Peng; Zhang, Yuchao; Liu, Yuantao; Chen, Jicui; Zong, Chen; Yu, Cong; Cui, Shang; Gao, Weina; Qin, Dandan; Sun, Wenchuan; Li, Xia; Wang, Xiangdong

    2015-12-01

    The role and mechanism of signal transducer and activator of transcription 5B (STAT5B) in adipogenesis remain unclear. In this study, our data showed that Males absent on the first (MOF) protein expression was increased during 3 T3-L1 preadipocytes differentiation accompanied with STAT5B expression increasing. Over-expression STAT5B enhanced MOF promoter trans-activation in HeLa cells. Mutagenesis assay and ChIP analysis exhibited that STAT5B was able to bind MOF promoter. Knocking-down STAT5B in 3 T3-L1 preadipocytes led to decreased expression of MOF, but resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid-binding protein 4 (Fabp4), which were important factors or enzymes for adipogenesis. We also found that knocking-down MOF in 3 T3-L1 preadipocytes resulted in increased expression of PPARγ, C/EBPα and Fabp4, which was in the same trend as STAT5B knocking-down. Over-expression MOF resulted in reduced promoter trans-activation activity of C/EBPα. These results suggest that STAT5B and MOF work as negative regulators in adipogenesis, and STAT5B modulates preadipocytes differentiation partially by regulating MOF expression.

  12. Reinstate the Damaged VEGF Signaling Pathway with VEGF-activating Transcription Factor

    Institute of Scientific and Technical Information of China (English)

    Yao-guo Yang; Heng Guan; Chang-wei Liu; Yong-jun Li

    2009-01-01

    Objective To investigate the role of vascular endothelial growth factor-activating transcriptional factor(VEGF-ATF)on the VEGF signaling pathway in diabetes mellitus.Methods Totally,20 C57BL/6 mice fed with high fat diet was induced into diabetes mellitus.Ten diabetes mellitus mice received a lower limb muscle injection with VEGF-ATF plasmid,and another ten were as control.VEGF-ATF is an engineered transcription factor designed to increase VEGF expression.Three days later,mice were sacrificed and the injected gastrocnemius was used for analysis.VEGF mRNA and protein expressions were examined by real-time PCR and ELISA respectively.VEGF receptor 2 mRNA expression was tested with RT-PCR.Phosphorylated Akt,Akt,endothelial nitric oxide synthase(eNOS),and phosphorylated eNOS were assessed by western blot.Results At 3 days post-injection,in mice with diabetes mellitus,VEGF gene transfer increased VEGF mRNA copies and VEGF protein expression in injected muscles compared with control;and reinstated the impaired VEGF signaling pathway with increasing the ratios of phosphorylated Akt/Akt and phosphorylated eNOS/eNOS.However,it did not affect the expression of VEGF receptor 2 mRNA.Conclusion Gene transfer with VEGF-ATF is able to reinstate the impaired VEGF downstream pathway,and potentially promote therapeutic angiogenesis in mice with diabetes mcllitus.

  13. Identification and characterization of jute LTR retrotransposons:: Their abundance, heterogeneity and transcriptional activity.

    Science.gov (United States)

    Ahmed, Salim; Shafiuddin, Md; Azam, Muhammad Shafiul; Islam, Md Shahidul; Ghosh, Ajit; Khan, Haseena

    2011-05-01

    Long Terminal Repeat (LTR) retrotransposons constitute a significant part of eukaryotic genomes and play an important role in genome evolution especially in plants. Jute is an important fiber crop with a large genome of 1,250 Mbps. This genome is still mostly unexplored. In this study we aimed at identifying and characterizing the LTR retrotransposons of jute with a view to understanding the jute genome better. In this study, the Reverse Transcriptase domain of Ty1-copia and Ty3-gypsy LTR retrotransposons of jute were amplified by degenerate primers and their expressions were examined by reverse transcription PCR. Copy numbers of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy elements were determined by dot blot analysis. Sequence analysis revealed higher heterogeneity among Ty1-copia retrotransposons than Ty3-gypsy and clustered each of them in three groups. Copy number of RT genes in Ty1-copia was found to be higher than that of Ty3-gypsy elements from dot blot hybridization. Cumulatively Ty1-copia and Ty3-gypsy may constitute around 19% of the jute genome where two groups of Ty1-copia were found to be transcriptionally active. Since the LTR retrotransposons constitute a large portion of jute genome, these findings imply the importance of these elements in the evolution of jute genome.

  14. Alteration of transcriptional networks in the entorhinal cortex after maternal immune activation and adolescent cannabinoid exposure.

    Science.gov (United States)

    Hollins, Sharon L; Zavitsanou, Katerina; Walker, Frederick Rohan; Cairns, Murray J

    2016-08-01

    Maternal immune activation (MIA) and adolescent cannabinoid exposure (ACE) have both been identified as major environmental risk factors for schizophrenia. We examined the effects of these two risk factors alone, and in combination, on gene expression during late adolescence. Pregnant rats were exposed to the viral infection mimic polyriboinosinic-polyribocytidylic acid (poly I:C) on gestational day (GD) 15. Adolescent offspring received daily injections of the cannabinoid HU210 for 14days starting on postnatal day (PND) 35. Gene expression was examined in the left entorhinal cortex (EC) using mRNA microarrays. We found prenatal treatment with poly I:C alone, or HU210 alone, produced relatively minor changes in gene expression. However, following combined treatments, offspring displayed significant changes in transcription. This dramatic and persistent alteration of transcriptional networks enriched with genes involved in neurotransmission, cellular signalling and schizophrenia, was associated with a corresponding perturbation in the expression of small non-coding microRNA (miRNA). These results suggest that a combination of environmental exposures during development leads to significant genomic remodeling that disrupts maturation of the EC and its associated circuitry with important implications as the potential antecedents of memory and learning deficits in schizophrenia and other neuropsychiatric disorders.

  15. Microchimeric fetal cells are recruited to maternal kidney following injury and activate collagen type I transcription.

    Science.gov (United States)

    Bou-Gharios, George; Amin, Farhana; Hill, Peter; Nakamura, Hiroyuki; Maxwell, Patrick; Fisk, Nicholas M

    2011-01-01

    Fetal cells enter the maternal circulation from the early first trimester of pregnancy, where they persist in tissue decades later. We investigated in mice whether fetal microchimeric cells (FMCs) can be detected in maternal kidney, and whether they play a role in kidney homeostasis. FMCs were identified in vivo in two models: one an adaptive model following unilateral nephrectomy, the other an injury via unilateral renal ischaemia reperfusion. Both models were carried out in mothers that had been mated with transgenic mice expressing luciferase transgene under the control of collagen type I, and had given birth to either 1 or 3 litters. FMCs were detected by Y-probe fluorescent in situ hybridization (FISH) and bioluminescence, and the cell number quantified by real-time polymerase chain reaction. In the adaptive model, the remaining kidney showed more cells by all 3 parameters compared with the nephrectomized kidney, while ischaemia reperfusion resulted in higher levels of FMC participation in injured compared to contralateral kidneys. Bioluminescence showed that FMCs switch on collagen type I transcription implicating mesenchymal lineage cells. After injury, Y-probe in situ hydridization was found mainly in the tubular epithelial network. Finally, we compared FMCs with bone marrow cells and found similar dynamics but altered distribution within the kidney. We conclude that FMCs (1) are long-term sequelae of pregnancy and (2) are recruited to the kidney as a result of injury or adaptation, where they activate the transcriptional machinery of matrix proteins.

  16. Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis.

    Science.gov (United States)

    Wassef, Michel; Rodilla, Veronica; Teissandier, Aurélie; Zeitouni, Bruno; Gruel, Nadege; Sadacca, Benjamin; Irondelle, Marie; Charruel, Margaux; Ducos, Bertrand; Michaud, Audrey; Caron, Matthieu; Marangoni, Elisabetta; Chavrier, Philippe; Le Tourneau, Christophe; Kamal, Maud; Pasmant, Eric; Vidaud, Michel; Servant, Nicolas; Reyal, Fabien; Meseure, Dider; Vincent-Salomon, Anne; Fre, Silvia; Margueron, Raphaël

    2015-12-15

    Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2's contribution to solid tumors with important therapeutic implications.

  17. Nε-lysine acetylation of a bacterial transcription factor inhibits Its DNA-binding activity.

    Directory of Open Access Journals (Sweden)

    Sandy Thao

    Full Text Available Evidence suggesting that eukaryotes and archaea use reversible N(ε-lysine (N(ε-Lys acetylation to modulate gene expression has been reported, but evidence for bacterial use of N(ε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs. We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat. Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD(+-dependent Sir2 (sirtuin-like protein deacetylase (CobB deacetylated acetylated RcsB (RcsB(Ac, demonstrating that N(ε-Lys acetylation of RcsB is reversible. Analysis of RcsB(Ac and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible N(ε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells.

  18. A do-it-yourself protocol for simple transcription activator-like effector assembly

    Directory of Open Access Journals (Sweden)

    Uhde-Stone Claudia

    2013-01-01

    Full Text Available Abstract Background TALEs (transcription activator-like effectors are powerful molecules that have broad applications in genetic and epigenetic manipulations. The simple design of TALEs, coupled with high binding predictability and specificity, is bringing genome engineering power to the standard molecular laboratory. Currently, however, custom TALE assembly is either costly or limited to few research centers, due to complicated assembly protocols, long set-up time and specific training requirements. Results We streamlined a Golden Gate-based method for custom TALE assembly. First, by providing ready-made, quality-controlled monomers, we eliminated the procedures for error-prone and time-consuming set-up. Second, we optimized the protocol toward a fast, two-day assembly of custom TALEs, based on four thermocycling reactions. Third, we increased the versatility for diverse downstream applications by providing series of vector sets to generate both TALENs (TALE nucleases and TALE-TFs (TALE-transcription factors under the control of different promoters. Finally, we validated our system by assembling a number of TALENs and TALE-TFs with DNA sequencing confirmation. We further demonstrated that an assembled TALE-TF was able to transactivate a luciferase reporter gene and a TALEN pair was able to cut its target. Conclusions We established and validated a do-it-yourself system that enables individual researchers to assemble TALENs and TALE-TFs within 2 days. The simplified TALE assembly combined with multiple choices of vectors will facilitate the broad use of TALE technology.

  19. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity.

    Science.gov (United States)

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N; Bradner, James E; Rabadan, Raul; Basu, Uttiya

    2015-05-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function.

  20. A two-subunit bacterial sigma-factor activates transcription in Bacillus subtilis.

    Science.gov (United States)

    MacLellan, Shawn R; Guariglia-Oropeza, Veronica; Gaballa, Ahmed; Helmann, John D

    2009-12-15

    The sigma-like factor YvrI and coregulator YvrHa activate transcription from a small set of conserved promoters in Bacillus subtilis. We report here that these two proteins independently contribute sigma-region 2 and sigma-region 4 functions to a holoenzyme-promoter DNA complex. YvrI binds RNA polymerase (RNAP) through a region 4 interaction with the beta-subunit flap domain and mediates specific promoter recognition but cannot initiate DNA melting at the -10 promoter element. Conversely, YvrHa possesses sequence similarity to a conserved core-binding motif in sigma-region 2 and binds to the N-terminal coiled-coil element in the RNAP beta'-subunit previously implicated in interaction with region 2 of sigma-factors. YvrHa plays an essential role in stabilizing the open complex and interacts specifically with the N-terminus of YvrI. Based on these results, we propose that YvrHa is situated in the transcription complex proximal to the -10 element of the promoter, whereas YvrI is responsible for -35 region recognition. This system presents an unusual example of a two-subunit bacterial sigma-factor.

  1. Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene.

    Science.gov (United States)

    Xia, Mengna; Malkaram, Sridhar A; Zempleni, Janos

    2013-11-01

    Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

  2. RAI1 transcription factor activity is impaired in mutants associated with Smith-Magenis Syndrome.

    Directory of Open Access Journals (Sweden)

    Paulina Carmona-Mora

    Full Text Available Smith-Magenis Syndrome (SMS is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1, located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1 and no ability to activate the transcription through an endogenous target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.

  3. RAI1 transcription factor activity is impaired in mutants associated with Smith-Magenis Syndrome.

    Science.gov (United States)

    Carmona-Mora, Paulina; Canales, Cesar P; Cao, Lei; Perez, Irene C; Srivastava, Anand K; Young, Juan I; Walz, Katherina

    2012-01-01

    Smith-Magenis Syndrome (SMS) is a complex genomic disorder mostly caused by the haploinsufficiency of the Retinoic Acid Induced 1 gene (RAI1), located in the chromosomal region 17p11.2. In a subset of SMS patients, heterozygous mutations in RAI1 are found. Here we investigate the molecular properties of these mutated forms and their relationship with the resulting phenotype. We compared the clinical phenotype of SMS patients carrying a mutation in RAI1 coding region either in the N-terminal or the C-terminal half of the protein and no significant differences were found. In order to study the molecular mechanism related to these two groups of RAI1 mutations first we analyzed those mutations that result in the truncated protein corresponding to the N-terminal half of RAI1 finding that they have cytoplasmic localization (in contrast to full length RAI1) and no ability to activate the transcription through an endogenous target: the BDNF enhancer. Similar results were found in lymphoblastoid cells derived from a SMS patient carrying RAI1 c.3103insC, where both mutant and wild type products of RAI1 were detected. The wild type form of RAI1 was found in the chromatin bound and nuclear matrix subcellular fractions while the mutant product was mainly cytoplasmic. In addition, missense mutations at the C-terminal half of RAI1 presented a correct nuclear localization but no activation of the endogenous target. Our results showed for the first time a correlation between RAI1 mutations and abnormal protein function plus they suggest that a reduction of total RAI1 transcription factor activity is at the heart of the SMS clinical presentation.

  4. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook;

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...

  5. Activation of Signal Transducer and Activator of Transcription 5 (STAT5) in Splenocyte Proliferation of Asthma Mice Induced by Ovalbumin

    Institute of Scientific and Technical Information of China (English)

    Guoping Li; Zhigang Liu; Peixing Ran; Jing Qiu; Nanshan Zhong

    2004-01-01

    To investigate the role of signal transducer and transcriptional activator 5 (STAT5) activated in ovalbumin (OVA)-induced splenocyte proliferation of asthma mice, an asthma mouse model was set up by intraperitoneal injection and aspiration of OVA with nebulizer. The proliferation of splenocytes isolated from the asthma mice was detected by [3H] thymidine incorporation. The phosphorytation of STAT5 was examined by Western blotting and STAT5-DNA binding was measured by electrophoretic mobility shift assay (EMSA). OVA could pronouncedly induce the splenocyte proliferation of asthma mice in a dose-dependent manner compared with control groups. Phosphorylation of STAT5 and STAT5-DNA binding were observed in splenocytes from asthma mice induced by OVA at 1 h and 3 h. These results indicated that STAT5 signal pathway played an important role in lymphocyte proliferation of asthma mice induced by OVA. Cellular & Molecular Immunology.2004;1(6):471-474.

  6. Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA

    OpenAIRE

    Kim, Jeong Hoon; Yang, Catherine K.; Stallcup, Michael R

    2006-01-01

    The coiled-coil coactivator (CoCoA) is a transcriptional coactivator for nuclear receptors and enhances nuclear receptor function by the interaction with the bHLH-PAS domain (AD3) of p160 coactivators. The C-terminal activation domain (AD) of CoCoA possesses strong transactivation activity and is required for the coactivator function of CoCoA with nuclear receptors. To understand how CoCoA AD transmits its activating signal to the transcription machinery, we defined specific subregions, amino...

  7. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    Science.gov (United States)

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene.

  8. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  9. Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Numamoto, Minori; Tagami, Shota; Ueda, Yusuke; Imabeppu, Yusuke; Sasano, Yu; Sugiyama, Minetaka; Maekawa, Hiromi; Harashima, Satoshi

    2015-08-01

    The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.

  10. A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    张宁; 徐永健; 张珍祥; 熊维宁

    2003-01-01

    To determine the feasibility of a nonradioactive electrophoresis mobility shift assay fordetecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus tar-get sequence of NF-κB were labled with DIG by terminal transferase. After nuclear protein stimula-ted with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDT C)electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, theywere electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemilu-minescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded spe-cifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method andthe activity of NF-κB in PMA group was more than that in PMA+PDTC group. It is suggestedthat detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, whichcan be performed in ordinary laboratories.

  11. USP10 Antagonizes c-Myc Transcriptional Activation through SIRT6 Stabilization to Suppress Tumor Formation

    Directory of Open Access Journals (Sweden)

    Zhenghong Lin

    2013-12-01

    Full Text Available The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.

  12. A structure-activity relationship study of small-molecule inhibitors of GLI1-mediated transcription.

    Science.gov (United States)

    Actis, Marcelo; Connelly, Michele C; Mayasundari, Anand; Punchihewa, Chandanamali; Fujii, Naoaki

    2011-01-01

    We have previously reported ketoprofen amide compounds as inhibitors of GLI1-mediated transcription, an essential down-stream element of the Hedgehog (Hh) pathway. These compounds inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with GLI1 and in Rh30 cells that endogenously overexpress GLI1. Here we have designed new derivatives of these compounds aiming to explore the structure-activation relationship (SAR). By replacing the ketone carbonyl group of the ketoprofen moiety with an ether, amide, sulfonamide, or sulfone, we found several new compounds that are equipotent to the ketoprofen amide compounds. Among them, sulfone 30 inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with GLI1 and in Rh30 cells that endogenously overexpress GLI1.

  13. KLF4 transcriptionally activates non-canonical WNT5A to control epithelial stratification.

    Science.gov (United States)

    Tetreault, Marie-Pier; Weinblatt, Daniel; Shaverdashvili, Khvaramze; Yang, Yizeng; Katz, Jonathan P

    2016-05-17

    Epithelial differentiation and stratification are essential for normal homeostasis, and disruption of these processes leads to both injury and cancer. The zinc-finger transciption factor KLF4 is a key driver of epithelial differentiation, yet the mechanisms and targets by which KLF4 controls differentiation are not well understood. Here, we define WNT5A, a non-canonical Wnt ligand implicated in epithelial differentiation, repair, and cancer, as a direct transcriptional target that is activated by KLF4 in squamous epithelial cells. Further, we demonstrate functionally that WNT5A mediates KLF4 control of epithelial differentiation and stratification, as treatment of keratinocytes with WNT5A rescues defective epithelial stratification resulting from KLF4 loss. Finally, we show that the small GTPase CDC42 is regulated by KLF4 in a WNT5A dependent manner. As such, we delineate a novel pathway for epithelial differentiation and stratification and define potential therapeutic targets for epithelial diseases.

  14. Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila.

    Science.gov (United States)

    Beumer, Kelly J; Trautman, Jonathan K; Christian, Michelle; Dahlem, Timothy J; Lake, Cathleen M; Hawley, R Scott; Grunwald, David J; Voytas, Daniel F; Carroll, Dana

    2013-10-03

    Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

  15. Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos.

    Science.gov (United States)

    Lei, Yong; Guo, Xiaogang; Deng, Yi; Chen, Yonglong; Zhao, Hui

    2013-05-10

    Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.

  16. Gene targeting in rats using transcription activator-like effector nucleases.

    Science.gov (United States)

    Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Thépenier, Virginie; Thinard, Reynald; Ouisse, Laure-Hélène; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-15

    The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

  17. Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast assembly.

    Science.gov (United States)

    Reyon, Deepak; Khayter, Cyd; Regan, Maureen R; Joung, J Keith; Sander, Jeffry D

    2012-10-01

    Engineered transcription activator-like effector nucleases (TALENs) are broadly useful tools for performing targeted genome editing in a wide variety of organisms and cell types including plants, zebrafish, C. elegans, rat, human somatic cells, and human pluripotent stem cells. Here we describe detailed protocols for the serial, hierarchical assembly of TALENs that require neither PCR nor specialized multi-fragment ligations and that can be implemented by any laboratory. These restriction enzyme and ligation (REAL)-based protocols can be practiced using plasmid libraries and user-friendly, Web-based software that both identifies target sites in sequences of interest and generates printable graphical guides that facilitate assembly of TALENs. With the described platform of reagents, protocols, and software, researchers can easily engineer multiple TALENs within 2 weeks using standard cloning techniques.

  18. Mutational analysis of the activator of late transcription, Alt , in the lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Hammer, Karin

    2007-01-01

    An activator protein, Alt, synthesized during the early state of lytic infection is required for transcription of the late operon in the lactococcal phage TP901-1. In order to identify amino acid residues in the Alt protein required for activation of the TP901-1 late promoter, Plate, hydroxylamin...

  19. A Global Genomic and Genetic Strategy to Predict Pathway Activation of Xenobiotic Responsive Transcription Factors in the Mouse Liver

    Science.gov (United States)

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

  20. Sumoylation of transcription factor Tec1 regulates signaling of mitogen-activated protein kinase pathways in yeast.

    Science.gov (United States)

    Wang, Yuqi; Abu Irqeba, Ameair; Ayalew, Mihretu; Suntay, Kristina

    2009-10-14

    Tec1 is a transcription factor in the yeast mitogen-activated protein kinase (MAPK) pathway that controls invasive growth. Previously we reported that a fraction of Tec1 protein is sumoylated on residue lysine 54 in normally growing cells. Here we describe regulation and functional consequences of Tec1 sumoylation. We found that activation of Kss1, the MAPK that directly activates Tec1, results in a decrease in Tec1 sumoylation and a concurrent increase of Tec1 transcriptional activity. Consistent with a role of sumoylation in inhibiting Tec1 activity, specifically increasing sumoylation of Tec1 by fusing it to the sumoylating enzyme Ubc9 leads to a dramatic decrease of Tec1 transcriptional activity. Invasive growth is also compromised in Tec1-Ubc9. In contrast, fusing sumoylation-site mutant Tec1, i.e., Tec1(K54R), to Ubc9 did not significantly alter transcriptional activation and had a less effect on invasive growth. Taken together, these findings provide evidence for regulated sumoylation as a mechanism to modulate the activity of Tec1 and validate Ubc9 fusion-directed sumoylation as a useful approach for studying protein sumoylation.

  1. Sumoylation of transcription factor Tec1 regulates signaling of mitogen-activated protein kinase pathways in yeast.

    Directory of Open Access Journals (Sweden)

    Yuqi Wang

    Full Text Available Tec1 is a transcription factor in the yeast mitogen-activated protein kinase (MAPK pathway that controls invasive growth. Previously we reported that a fraction of Tec1 protein is sumoylated on residue lysine 54 in normally growing cells. Here we describe regulation and functional consequences of Tec1 sumoylation. We found that activation of Kss1, the MAPK that directly activates Tec1, results in a decrease in Tec1 sumoylation and a concurrent increase of Tec1 transcriptional activity. Consistent with a role of sumoylation in inhibiting Tec1 activity, specifically increasing sumoylation of Tec1 by fusing it to the sumoylating enzyme Ubc9 leads to a dramatic decrease of Tec1 transcriptional activity. Invasive growth is also compromised in Tec1-Ubc9. In contrast, fusing sumoylation-site mutant Tec1, i.e., Tec1(K54R, to Ubc9 did not significantly alter transcriptional activation and had a less effect on invasive growth. Taken together, these findings provide evidence for regulated sumoylation as a mechanism to modulate the activity of Tec1 and validate Ubc9 fusion-directed sumoylation as a useful approach for studying protein sumoylation.

  2. High-resolution mapping and transcriptional activity analysis of chicken centromere sequences on giant lampbrush chromosomes.

    Science.gov (United States)

    Krasikova, Alla; Fukagawa, Tatsuo; Zlotina, Anna

    2012-12-01

    Exploration into morphofunctional organisation of centromere DNA sequences is important for understanding the mechanisms of kinetochore specification and assembly. In-depth epigenetic analysis of DNA fragments associated with centromeric nucleosome proteins has demonstrated unique features of centromere organisation in chicken karyotype: there are both mature centromeres, which comprise chromosome-specific homogeneous arrays of tandem repeats, and recently evolved primitive centromeres, which consist of non-tandemly organised DNA sequences. In this work, we describe the arrangement and transcriptional activity of chicken centromere repeats for Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 and non-repetitive centromere sequences of chromosomes 5, 27, and Z using highly elongated lampbrush chromosomes, which are characteristic of the diplotene stage of oogenesis. The degree of chromatin packaging and fine spatial organisations of tandemly repetitive and non-tandemly repetitive centromeric sequences significantly differ at the lampbrush stage. Using DNA/RNA FISH, we have demonstrated that during the lampbrush stage, DNA sequences are transcribed within the centromere regions of chromosomes that lack centromere-specific tandem repeats. In contrast, chromosome-specific centromeric repeats Cen1, Cen2, Cen3, Cen4, Cen7, Cen8, and Cen11 do not demonstrate any transcriptional activity during the lampbrush stage. In addition, we found that CNM repeat cluster localises adjacent to non-repetitive centromeric sequences in chicken microchromosome 27 indicating that centromere region in this chromosome is repeat-rich. Cross-species FISH allowed localisation of the sequences homologous to centromeric DNA of chicken chromosomes 5 and 27 in centromere regions of quail orthologous chromosomes.

  3. Mechanisms of transcriptional activation of the mouse claudin-5 promoter by estrogen receptor alpha and beta.

    Science.gov (United States)

    Burek, Malgorzata; Steinberg, Katrin; Förster, Carola Y

    2014-07-01

    Claudin-5 is an integral membrane protein and a critical component of endothelial tight junctions that control paracellular permeability. Claudin-5 is expressed at high levels in the brain vascular endothelium. Estrogens have multiple effects on vascular physiology and function. The biological actions of estrogens are mediated by two different estrogen receptor (ER) subtypes, ER alpha and ER beta. Estrogens have beneficial effects in several vascular disorders. Recently we have cloned and characterized a murine claudin-5 promoter and demonstrated 17beta-estradiol (E2)-mediated regulation of claudin-5 in brain and heart microvascular endothelium on promoter, mRNA and protein level. Sequence analysis revealed a putative estrogen response element (ERE) and a putative Sp1 transcription factor binding site in the claudin-5 promoter. The aim of the present study was to further characterize the estrogen-responsive elements of claudin-5 promoter. First, we introduced point mutations in ERE or Sp1 site in -500/+111 or in Sp1 site of -268/+111 claudin-5 promoter construct, respectively. Basal and E2-mediated transcriptional activation of mutated constructs was abrogated in the luciferase reporter gene assay. Next, we examined whether estrogen receptor subtypes bind to the claudin-5 promoter region. For this purpose we performed chromatin immunoprecipitation assays using anti-estrogen receptor antibodies and cellular lysates of E2-treated endothelial cells followed by quantitative PCR analysis. We show enrichment of claudin-5 promoter fragments containing the ERE- and Sp1-binding site in immunoprecipitates after E2 treatment. Finally, in a gel mobility shift assay, we demonstrated DNA-protein interaction of both ER subtypes at ERE. In summary, this study provides evidence that both a non-consensus ERE and a Sp1 site in the claudin-5 promoter are functional and necessary for the basal and E2-mediated activation of the promoter.

  4. MicroRNA-214 Suppresses Gluconeogenesis by Targeting Activating Transcriptional Factor 4*

    Science.gov (United States)

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-01-01

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. PMID:25657009

  5. Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma

    Directory of Open Access Journals (Sweden)

    Lazarus Ross

    2005-06-01

    Full Text Available Abstract Background Identifying genetic determinants for lung function is important in providing insight into the pathophysiology of asthma. Signal transducer and activator of transcription 3 is a transcription factor latent in the cytoplasm; the gene (STAT3 is activated by a wide range of cytokines, and may play a role in lung development and asthma pathogenesis. Methods We genotyped six single nucleotide polymorphisms (SNPs in the STAT3 gene in a cohort of 401 Caucasian adult asthmatics. The associations between each SNP and forced expiratory volume in 1 second (FEV1, as a percent of predicted, at the baseline exam were tested using multiple linear regression models. Longitudinal analyses involving repeated measures of FEV1 were conducted with mixed linear models. Haplotype analyses were conducted using imputed haplotypes. We completed a second association study by genotyping the same six polymorphisms in a cohort of 652 Caucasian children with asthma. Results We found that three polymorphisms were significantly associated with baseline FEV1: homozygotes for the minor alleles of each polymorphism had lower FEV1 than homozygotes for the major alleles. Moreover, these associations persisted when we performed an analysis on repeated measures of FEV1 over 8 weeks. A haplotypic analysis based on the six polymorphisms indicated that two haplotypes were associated with baseline FEV1. Among the childhood asthmatics, one polymorphism was associated with both baseline FEV1 and the repeated measures of FEV1 over 4 years. Conclusion Our results indicate that genetic variants in STAT3, independent of asthma treatment, are determinants of FEV1 in both adults and children with asthma, and suggest that STAT3 may participate in inflammatory pathways that have an impact on level of lung function.

  6. Involvement of tristetraprolin in transcriptional activation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase by insulin

    Energy Technology Data Exchange (ETDEWEB)

    Ness, Gene C., E-mail: gness@hsc.usf.edu [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States); Edelman, Jeffrey L.; Brooks, Patricia A. [Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612 (United States)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer siRNAs to tristetraprolin blocks transcription of HMGR in vivo in rat liver. Black-Right-Pointing-Pointer siRNAs to tristetraprolin inhibits insulin activation of HMGR transcription. Black-Right-Pointing-Pointer Insulin acts to rapidly increase tristetraprolin in liver nuclear extracts. -- Abstract: Several AU-rich RNA binding element (ARE) proteins were investigated for their possible effects on transcription of hepatic 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMGR) in normal rats. Using in vivo electroporation, four different siRNAs to each ARE protein were introduced together with HMGR promoter (-325 to +20) luciferase construct and compared to saline controls. All four siRNAs to tristetraprolin (TTP) completely eliminated transcription from the HMGR promoter construct. Since insulin acts to rapidly increase hepatic HMGR transcription, the effect of TTP siRNA on induction by insulin was tested. The 3-fold stimulation by insulin was eliminated by this treatment. In comparison, siRNA to AU RNA binding protein/enoyl coenzyme A hydratase (AUH) had no effect. These findings indicate a role for TTP in the insulin-mediated activation of hepatic HMGR transcription.

  7. Role of hypoxia-inducible factor-1 in transcriptional activation of ceruloplasmin by iron deficiency

    Science.gov (United States)

    Mukhopadhyay, C. K.; Mazumder, B.; Fox, P. L.

    2000-01-01

    A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta, as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these

  8. Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ibtissam Youlyouz

    2002-01-01

    Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

  9. Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival

    Directory of Open Access Journals (Sweden)

    Hsieh Fu-Chuan

    2008-10-01

    Full Text Available Abstract Background Constitutive activation of signal transducer and activator of transcription 3 (Stat3 signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer. Results We found that elevated Stat3 phosphorylation in 19 of 100 (19% bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC. The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin and a cell cycle regulating gene (cyclin D1 was associated with the cell growth inhibition and apoptosis. Conclusion These results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

  10. BAF Complex Is Closely Related to and Interacts with NF1/CTF and RNA Polymerase Ⅱ in Gene Transcriptional Activation

    Institute of Scientific and Technical Information of China (English)

    Li-Hui ZHAO; Xue-Qing BA; Xiao-Guang WANG; Xiao-Juan ZHU; Li WANG; Xian-Lu ZENG

    2005-01-01

    Brg- or hBrm-associated factor (BAF) complexes, a chromatin-remodeling complex family of mammalian cells, facilitate transcriptional activity by remodeling nucleosome structure. Brg 1 is the core subunit of Brg-associated factor complexes. In the present study, we investigated the spatial relationship between Brg1 and nuclear factor 1 (NF1/CTF) and RNA polymerase Ⅱ (RNAP Ⅱ) upon gene transcriptional activation in vivo by employing immuno-gold labeling. The data showed that Brg1 was closely co-localized with NF1/CTF and RNAP Ⅱ in HeLa cells. Moreover, the co-immunoprecipitation assay further revealed that Brg1 can be isolated together with NF1/CTF and RNAP Ⅱ in the ConA-stimulated, but not the resting,T lymphocyte. The combined results suggested that BAF complexes can interact with NF1/CTF and RNAP Ⅱ, and this interaction is closely dependent on the activation of gene transcription.

  11. CMYB1 Encoding a MYB Transcriptional Activator Is Involved in Abiotic Stress and Circadian Rhythm in Rice

    Directory of Open Access Journals (Sweden)

    Min Duan

    2014-01-01

    Full Text Available Through analysis of cold-induced transcriptome, a novel gene encoding a putative MYB transcription factor was isolated and designated Cold induced MYB 1 (CMYB1. Tissue-specific gene expression analysis revealed that CMYB1 was highly expressed in rice stems and nodes. qRT-PCR assay indicated that CMYB1 was dramatically induced by cold stress (>100-folds and induced by exogenous ABA and osmotic stress. Interestingly, CMYB1 showed rhythmic expression profile in rice leaves at different developmental stages. Subcellular localization assay suggested that CMYB1-GFP (green fluorescent protein fusion protein was localized in the nuclei. Moreover, CMYB1 exhibited the transcriptional activation activity when transiently expressed in rice protoplast cells. Taken together, CMYB1 probably functions as a transcriptional activator in mediating stress and rhythm responsive gene expression in rice.

  12. Novel Principles of Gamma-Retroviral Insertional Transcription Activation in Murine Leukemia Virus-induced End-stage Tumors

    DEFF Research Database (Denmark)

    Sokol, Martin; Wabl, Matthias; Rius Ruiz, Irene;

    2014-01-01

    Background Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has...... been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced...... than 2,000 mice was examined. Results We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located...

  13. Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

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    Irma Eloisa Monroy-Muñoz

    2015-01-01

    Full Text Available Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD using the High Resolution Melting (HRM method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up.

  14. Transcriptional activation by pRB and its coordination with SWI/SNF recruitment.

    Science.gov (United States)

    Flowers, Stephen; Beck, George R; Moran, Elizabeth

    2010-11-01

    A central question in cancer biology is why most tumor susceptibility genes are linked with only limited types of cancer. Human germ-line mutation of the retinoblastoma susceptibility gene Rb1 is closely linked with just retinoblastoma and osteosarcoma, although the gene is universally expressed. Functional analysis of pRB and its close relatives, p107 and p130, has largely focused on their roles in repression of proliferation across all tissue types, but genetic evidence indicates an active requirement for pRB in osteoblast differentiation that correlates more directly with osteosarcoma susceptibility. Still, potential promoter targets of pRB and its role in normally differentiating osteoblasts remain insufficiently characterized. Here, an early marker of osteoblast differentiation, alkaline phosphatase, is identified as a direct promoter activation target of pRB. One role of pRB on this promoter is to displace the histone lysine demethylase KDM5A, thereby favoring trimethylation of H3K4, a promoter activation mark. A major new aspect of pRB-mediated transcriptional activation revealed in this promoter analysis is its role in recruitment of an activating SWI/SNF chromatin-remodeling complex. SWI/SNF is a critical coordinator of tissue-specific gene expression. In osteoblasts, SWI/SNF complexes containing the BRM ATPase repress osteoblast-specific genes to maintain the precursor state, whereas the alternative ATPase BRG1 distinguishes an activating SWI/SNF complex necessary for RNA polymerase-II recruitment. A switch from BRM to BRG1 on the alkaline phosphatase promoter marks the onset of differentiation and is accomplished in a precise two-step mechanism. Dissociation of BRM-containing SWI/SNF depends on p300, and association of BRG1-containing SWI/SNF depends on pRB.

  15. Non-additive transcriptional profiles underlie dikaryotic superiority in Pleurotus ostreatus laccase activity.

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    Raúl Castanera

    Full Text Available BACKGROUND: The basidiomycete Pleurotus ostreatus is an efficient producer of laccases, a group of enzymes appreciated for their use in multiple industrial processes. The aim of this study was to reveal the molecular basis of the superiority of laccase production by dikaryotic strains compared to their parental monokaryons. METHODOLOGY/PRINCIPAL FINDINGS: We bred and studied a set of dikaryotic strains starting from a meiotic population of monokaryons. We then completely characterised the laccase allelic composition, the laccase gene expression and activity profiles in the dikaryotic strain N001, in two of its meiotic full-sib monokaryons and in the dikaryon formed from their mating. CONCLUSIONS/SIGNIFICANCE: Our results suggested that the dikaryotic superiority observed in laccase activity was due to non-additive transcriptional increases in lacc6 and lacc10 genes. Furthermore, the expression of these genes was divergent in glucose- vs. lignocellulose-supplemented media and was highly correlated to the detected extracellular laccase activity. Moreover, the expression profile of lacc2 in the dikaryotic strains was affected by its allelic composition, indicating a putative single locus heterozygous advantage.

  16. Characterization of transcriptional activation and DNA-binding functions in the hinge region of the vitamin D receptor.

    Science.gov (United States)

    Shaffer, Paul L; McDonnell, Donald P; Gewirth, Daniel T

    2005-02-22

    The vitamin D receptor (VDR) is a ligand-responsive transcription factor that forms active, heterodimeric complexes with the 9-cis retinoic acid receptor (RXR) on vitamin D response elements (VDREs). Both proteins consist of an N-terminal DNA-binding domain, a C-terminal ligand-binding domain, and an intervening hinge region. The length requirements of the hinge for both transcriptional regulation and DNA binding have not been studied to date for any member of the steroid hormone superfamily. We have generated a series of internal deletion mutants of the VDR hinge and found that deletion of as few as five amino acids from the C-terminus of the hinge significantly reduces transcriptional activation in vivo. Replacing deleted residues in the C-terminus of the hinge with alanines restored activity, indicating that this section of the hinge acts as a sequence-independent spacer. The hinge region of VDR forms a long helix, and the geometric consequences of this structure may explain the requirement of the hinge region for transcriptional activity. Interestingly, all of the deletion mutants, even those that do not activate transcription, bind VDREs with equal and high affinity, indicating that the defect in these mutants is not their ability to bind VDREs. In contrast to VDR, constructs of RXR containing deletions of up to 14 amino acids in the hinge region exhibit near wild-type transcriptional activity. The ability to delete more of the RXR hinge may be related to the additional plasticity required by its role as the common heterodimer partner for nuclear receptors on differing DNA elements.

  17. The Runx transcriptional co-activator, CBFβ, is essential for invasion of breast cancer cells

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    Lopez-Camacho Cesar

    2010-06-01

    Full Text Available Abstract Background The transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBFβ is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBFβ is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBFβ-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBFβ. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBFβ expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBFβ. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis. Results We show that CBFβ is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBFβ contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBFβ is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBFβ is recruited to both the Osteopontin and the Galectin-3 promoters. Conclusions CBFβ is expressed in metastatic breast cancer cells and is essential for cell invasion. CBFβ is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBFβ is essential for invasion, not all Runx2-target genes require CBFβ. We conclude that CBFβ is required for a subset of Runx2-target genes

  18. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data

    Science.gov (United States)

    Berchtold, Evi; Csaba, Gergely; Zimmer, Ralf

    2016-01-01

    Several methods predict activity changes of transcription factors (TFs) from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score), which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal) i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score. PMID:27723775

  19. Molecular recognition: monomer of the yeast transcriptional activator GCN4 recognizes its dimer DNA binding target sites specifically

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    It is widely believed that dimerization is a requirement for the yeast transcriptional activator GCN4 to recognize its specific DNA target sites. We used the basic region (226-252) of the yeast transcriptional activator GCN4, as both a monomeric peptide and a disulfide-linked dimer to investigate the interaction of the peptides with the DNA target sites AP-1 and CRE. CD and ITC experiments indicate that although the monomeric peptide GCN4-M has a weaker affinity with the DNA relative to the disulfide-linked dimer peptide GCN4-D, it recognizes AP-1 and CRE target sites specifically.

  20. RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13.

    Directory of Open Access Journals (Sweden)

    Ila Datar

    Full Text Available Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP

  1. Ribbon regulates morphogenesis of the Drosophila embryonic salivary gland through transcriptional activation and repression.

    Science.gov (United States)

    Loganathan, Rajprasad; Lee, Joslynn S; Wells, Michael B; Grevengoed, Elizabeth; Slattery, Matthew; Andrew, Deborah J

    2016-01-01

    Transcription factors affect spatiotemporal patterns of gene expression often regulating multiple aspects of tissue morphogenesis, including cell-type specification, cell proliferation, cell death, cell polarity, cell shape, cell arrangement and cell migration. In this work, we describe a distinct role for Ribbon (Rib) in controlling cell shape/volume increases during elongation of the Drosophila salivary gland (SG). Notably, the morphogenetic changes in rib mutants occurred without effects on general SG cell attributes such as specification, proliferation and apoptosis. Moreover, the changes in cell shape/volume in rib mutants occurred without compromising epithelial-specific morphological attributes such as apicobasal polarity and junctional integrity. To identify the genes regulated by Rib, we performed ChIP-seq analysis in embryos driving expression of GFP-tagged Rib specifically in the SGs. To learn if the Rib binding sites identified in the ChIP-seq analysis were linked to changes in gene expression, we performed microarray analysis comparing RNA samples from age-matched wild-type and rib null embryos. From the superposed ChIP-seq and microarray gene expression data, we identified 60 genomic sites bound by Rib likely to regulate SG-specific gene expression. We confirmed several of the identified Rib targets by qRT-pCR and/or in situ hybridization. Our results indicate that Rib regulates cell growth and tissue shape in the Drosophila salivary gland via a diverse array of targets through both transcriptional activation and repression. Furthermore, our results suggest that autoregulation of rib expression may be a key component of the SG morphogenetic gene network.

  2. Signal Transducer and Activator of Transcription 3 (STAT3 and Trophoblast Invasion

    Directory of Open Access Journals (Sweden)

    Fitzgerald JS

    2007-01-01

    Full Text Available Human trophoblast cells have the fascinating property of physiological invasiveness into allogenic tissue. The underlying mechanisms, such as extra- and intracellular signalling, are very similar to those abused by a variety of tumours. The main contrasting feature to cancerous cells is the very fundamental ability of trophoblasts to auto-regulate invasion with respect to time and space. Trophoblast cells start invasion into the decidua very shortly after implantation, which approaches a maximum during the first trimester of gestation period. During this period of time, several cytokines from cells of different maternal origin, including NK cells, dendritic cells, stroma cells and endothelial cells, are present which, analogous to the situation in tumours, have the potential to trigger and enhance invasion, migration and proliferation of trophoblast cells. These mainly include interleukin-6 (IL-6, IL-11, Leukaemia Inhibitory Factor (LIF, Hepatocyte Growth Factor (HGF and Insulin-like Growth Factors (IGF. Cytokines, upon binding to their specific receptors present on the trophoblast cells, trigger several intracellular signalling cascades. One of these signalling pathways is the Janus Kinase (Jak/Signal Transducers and Activators of Transcription (STAT pathway. As recent studies have shown, the tyrosine phosphorylated form of STAT3 is a major inducer of invasiveness which mainly takes place upon binding of LIF to its receptor. For autoregulation of signals, STAT3 induces the transcription of Suppressor of Cytokine Signalling 3 (SOCS3. The balance between STAT3 and SOCS3 may be argued as one of the main tuners of trophoblast invasion for successful implantation. Disturbances in this balance may lead to serious complications like cancer and implantation failure.

  3. The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Vidal M.S.

    2002-01-01

    Full Text Available Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22. Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

  4. The Drosophila Transcription Factors Tinman and Pannier Activate and Collaborate with Myocyte Enhancer Factor-2 to Promote Heart Cell Fate.

    Directory of Open Access Journals (Sweden)

    TyAnna L Lovato

    Full Text Available Expression of the MADS domain transcription factor Myocyte Enhancer Factor 2 (MEF2 is regulated by numerous and overlapping enhancers which tightly control its transcription in the mesoderm. To understand how Mef2 expression is controlled in the heart, we identified a late stage Mef2 cardiac enhancer that is active in all heart cells beginning at stage 14 of embryonic development. This enhancer is regulated by the NK-homeodomain transcription factor Tinman, and the GATA transcription factor Pannier through both direct and indirect interactions with the enhancer. Since Tinman, Pannier and MEF2 are evolutionarily conserved from Drosophila to vertebrates, and since their vertebrate homologs can convert mouse fibroblast cells to cardiomyocytes in different activator cocktails, we tested whether over-expression of these three factors in vivo could ectopically activate known cardiac marker genes. We found that mesodermal over-expression of Tinman and Pannier resulted in approximately 20% of embryos with ectopic Hand and Sulphonylurea receptor (Sur expression. By adding MEF2 alongside Tinman and Pannier, a dramatic expansion in the expression of Hand and Sur was observed in almost all embryos analyzed. Two additional cardiac markers were also expanded in their expression. Our results demonstrate the ability to initiate ectopic cardiac fate in vivo by the combination of only three members of the conserved Drosophila cardiac transcription network, and provide an opportunity for this genetic model system to be used to dissect the mechanisms of cardiac specification.

  5. Conserved region 2.1 of Escherichia coli heat shock transcription factor sigma32 is required for modulating both metabolic stability and transcriptional activity.

    Science.gov (United States)

    Horikoshi, Mina; Yura, Takashi; Tsuchimoto, Sachie; Fukumori, Yoshihiro; Kanemori, Masaaki

    2004-11-01

    Escherichia coli heat shock transcription factor sigma32 is rapidly degraded in vivo, with a half-life of about 1 min. A set of proteins that includes the DnaK chaperone team (DnaK, DnaJ, GrpE) and ATP-dependent proteases (FtsH, HslUV, etc.) are involved in degradation of sigma32. To gain further insight into the regulation of sigma32 stability, we isolated sigma32 mutants that were markedly stabilized. Many of the mutants had amino acid substitutions in the N-terminal half (residues 47 to 55) of region 2.1, a region highly conserved among bacterial sigma factors. The half-lives ranged from about 2-fold to more than 10-fold longer than that of the wild-type protein. Besides greater stability, the levels of heat shock proteins, such as DnaK and GroEL, increased in cells producing stable sigma32. Detailed analysis showed that some stable sigma32 mutants have higher transcriptional activity than the wild type. These results indicate that the N-terminal half of region 2.1 is required for modulating both metabolic stability and the activity of sigma32. The evidence suggests that sigma32 stabilization does not result from an elevated affinity for core RNA polymerase. Region 2.1 may, therefore, be involved in interactions with the proteolytic machinery, including molecular chaperones.

  6. Evidence that intramolecular interactions are involved in masking the activation domain of transcriptional activator Leu3p.

    Science.gov (United States)

    Wang, D; Hu, Y; Zheng, F; Zhou, K; Kohlhaw, G B

    1997-08-01

    The Leu3 protein of Saccharomyces cerevisiae regulates the expression of genes involved in branched chain amino acid biosynthesis and in ammonia assimilation. It is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis. In the presence of alpha-isopropylmalate, Leu3p is a transcriptional activator. In the absence of the signal molecule, the activation domain is masked, and Leu3p acts as a repressor. The recent discovery that Leu3p retains its regulatory properties when expressed in mammalian cells (Guo, H., and Kohlhaw, G. B. (1996) FEBS Lett. 390, 191-195) suggests that masking and unmasking of the activation domain occur without the participation of auxiliary proteins. Here we present experimental support for this notion and address the mechanism of masking. We show that modulation of Leu3p is exceedingly sensitive to mutations in the activation domain. An activation domain double mutant (D872N/D874N; designated Leu3-dd) was constructed that has the characteristics of a permanently masked activator. Using separately expressed segments containing either the DNA binding domain-middle region or the activation domain of wild type Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide direct evidence for alpha-isopropylmalate-dependent interaction between these segments. Finally, we use the phenotype of Leu3-dd-containing cells (slow growth in the absence of added leucine) to select for suppressor mutations that map to the middle region of Leu3-dd. The properties of nine such suppressors further support the idea that masking is an intramolecular process and suggest a means for mapping the surface involved in masking.

  7. Arabidopsis sigma factor binding proteins are activators of the WRKY33 transcription factor in plant defense.

    Science.gov (United States)

    Lai, Zhibing; Li, Ying; Wang, Fei; Cheng, Yuan; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2011-10-01

    Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif-containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens.

  8. Drosophila SAF-B links the nuclear matrix, chromosomes, and transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Catalina Alfonso-Parra

    Full Text Available Induction of gene expression is correlated with alterations in nuclear organization, including proximity to other active genes, to the nuclear cortex, and to cytologically distinct domains of the nucleus. Chromosomes are tethered to the insoluble nuclear scaffold/matrix through interaction with Scaffold/Matrix Attachment Region (SAR/MAR binding proteins. Identification and characterization of proteins involved in establishing or maintaining chromosome-scaffold interactions is necessary to understand how the nucleus is organized and how dynamic changes in attachment are correlated with alterations in gene expression. We identified and characterized one such scaffold attachment factor, a Drosophila homolog of mammalian SAF-B. The large nuclei and chromosomes of Drosophila have allowed us to show that SAF-B inhabits distinct subnuclear compartments, forms weblike continua in nuclei of salivary glands, and interacts with discrete chromosomal loci in interphase nuclei. These interactions appear mediated either by DNA-protein interactions, or through RNA-protein interactions that can be altered during changes in gene expression programs. Extraction of soluble nuclear proteins and DNA leaves SAF-B intact, showing that this scaffold/matrix-attachment protein is a durable component of the nuclear matrix. Together, we have shown that SAF-B links the nuclear scaffold, chromosomes, and transcriptional activity.

  9. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs).

    Science.gov (United States)

    Moore, Finola E; Reyon, Deepak; Sander, Jeffry D; Martinez, Sarah A; Blackburn, Jessica S; Khayter, Cyd; Ramirez, Cherie L; Joung, J Keith; Langenau, David M

    2012-01-01

    Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  10. Rapid and efficient assembly of transcription activator-like effector genes by USER cloning.

    Science.gov (United States)

    Wang, Song; Li, Wei; Wang, Shuo; Hu, Baoyang

    2014-06-20

    Transcription activator-like effectors (TALEs) that were related to bacteria immune system have lately been employed in a promising approach of precise gene targeting. Because of the repetitive characteristics of TALEs, existing TALE assembly methods are either very complicated, time-consuming, or too tricky to be handled in common labs. Here, we reported a rapid, efficient and easy method for TALE assembly. This method takes advantage of uracil-specific excision reagent (USER), an enzyme that can cleave DNA constructs and create long, unique single-strand DNA overhangs. Upon USER treatment, the overhangs on each individual TALE repeat unit can be rejoined hierarchically to form pentamers in a ligation-independent manner. Eventually, three pentamers are assembled into a full TALE construct by Golden Gate cloning. TALE nucleases (TALENs) generated with this method exhibit high genome-editing activity in human cells such as HEK293FT cells. Using this method, we have successfully synthesized three TALEN pairs targeting endogenous Tet1 locus, and proved that all can specifically target Tet1 gene, though in various degree. Comparing to other methods of TALEN assembly, this one is much less labor intensive and fairly faster, and positive clones can be obtained at high efficiency within only two days. We thus contribute to an easier approach for effective TALENs synthesis, which may highly facilitate the wide application of TALEN technology in genome editing, especially for human cells that require precise targeting.

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

    Science.gov (United States)

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien; Thomas, Séverine; Stella, Stefano; Maréchal, Alan; Langevin, Stéphanie; Benomari, Nassima; Bertonati, Claudia; Silva, George H; Daboussi, Fayza; Epinat, Jean-Charles; Montoya, Guillermo; Duclert, Aymeric; Duchateau, Philippe

    2014-04-01

    A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.

  12. Transcription activator-like effector nucleases (TALEN-mediated targeted DNA Insertion in potato plants

    Directory of Open Access Journals (Sweden)

    Adrienne Forsyth

    2016-10-01

    Full Text Available Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines TALEN-mediated induction of double strand breaks (DSBs and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

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

    Science.gov (United States)

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

    2013-06-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

  14. Low ozone concentrations stimulate cytoskeletal organization, mitochondrial activity and nuclear transcription

    Directory of Open Access Journals (Sweden)

    M. Costanzo

    2015-04-01

    Full Text Available Ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone concentrations and used in medicine as an alternative/adjuvant treatment for different diseases. However, the cellular mechanisms accounting for the positive effects of mild ozonization are still largely unexplored. To this aim, in the present study the effects of low ozone concentrations (1 to 20 µg O3/mL O2 on structural and functional cell features have been investigated in vitro by using morphological, morphometrical, cytochemical and immunocytochemical techniques at bright field, fluorescence and transmission electron microscopy. Cells exposed to pure O2 or air served as controls. The results demonstrated that the effects of ozoneadministration are dependent on gas concentration, and the cytoskeletal organization, mitochondrial activity and nuclear transcription may be differently affected. This suggests that, to ensure effective and permanent metabolic cell activation, ozone treatments should take into account the cytological and cytokinetic features of the different tissues. 

  15. ATP-dependent transcriptional activation by bacterial PspF AAA+protein.

    Science.gov (United States)

    Schumacher, Jörg; Zhang, Xiaodong; Jones, Susan; Bordes, Patricia; Buck, Martin

    2004-05-14

    Transcription activation by bacterial sigma(54)-dependent enhancer-binding proteins (EBPs) requires their tri-nucleotide hydrolysis to restructure the sigma(54) RNA polymerase (RNAP). EBPs share sequence similarity with guanine nucleotide binding-proteins and ATPases associated with various cellular activities (AAA) proteins, especially in the mononucleotide binding P-loop fold. Using the phage shock protein F (PspF) EBP, we identify P-loop residues responsible for nucleotide binding and hydrolysis, consistent with their roles in other P-loop NTPases. We show the refined low-resolution structure of an EBP, PspF, revealing a hexameric ring organisation characteristic of AAA proteins. Functioning of EBPs involves ATP binding, higher oligomer formation and ATP hydrolysis coupled to the restructuring of the RNAP. This is thought to be a highly coordinated multi-step process, but the nucleotide-driven mechanism of oligomerisation and ATP hydrolysis is little understood. Our kinetic and structural data strongly suggest that three PspF dimers assemble to form a hexamer upon nucleotide binding. During the ATP hydrolysis cycle, both ATP and ADP are bound to oligomeric PspF, in line with a sequential hydrolysis cycle. We identify a putative R-finger, and show its involvement in ATP hydrolysis. Substitution of this arginine residue results in nucleotide-independent formation of hexameric rings, structurally linking the putative R-finger and, by inference, a specific nucleotide interaction to the control of PspF oligomerisation.

  16. Krebs cycle function is required for activation of the Spo0A transcription factor in Bacillus subtilis.

    OpenAIRE

    Ireton, K.; Jin, S.; Grossman, A D; Sonenshein, A L

    1995-01-01

    Expression of genes early during sporulation in Bacillus subtilis requires the activity of the transcription factor encoded by spo0A. The active, phosphorylated form of Spo0A is produced through the action of a multicomponent pathway, the phosphorelay. A mutant defective in the first three enzymes of the Krebs citric acid cycle was unable to express early sporulation genes, apparently because of a failure to activate the phosphorelay. Cells that produce an altered Spo0A protein that can be ph...

  17. Possible Involvement of Nitric Oxide and Reactive Oxygen Species in Glucose Deprivation-Induced Activation of Transcription Factor Rst2

    OpenAIRE

    Toshiaki Kato; Xin Zhou; Yan Ma

    2013-01-01

    Glucose is one of the most important sources of cellular nutrition and glucose deprivation induces various cellular responses. In Schizosaccharomyces pombe, zinc finger protein Rst2 is activated upon glucose deprivation, and regulates gene expression via the STREP (stress response element of Schizosaccharomyces pombe) motif. However, the activation mechanism of Rst2 is not fully understood. We monitored Rst2 transcriptional activity in living cells using a Renilla luciferase reporter system. ...

  18. Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Zhao, Shaomin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Song, Langying [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Manyuan [School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Jiao, Kai, E-mail: kjiao@uab.edu [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2013-11-29

    Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, the activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.

  19. Transcription activator-like effector nucleases enable efficient plant genome engineering.

    Science.gov (United States)

    Zhang, Yong; Zhang, Feng; Li, Xiaohong; Baller, Joshua A; Qi, Yiping; Starker, Colby G; Bogdanove, Adam J; Voytas, Daniel F

    2013-01-01

    The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here, we describe methods for the targeted modification of plant genomes using transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.

  20. Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Chronic Lymphocytic Leukemia Cells

    Science.gov (United States)

    Liu, Zhiming; Hazan-Halevy, Inbal; Harris, David M.; Li, Ping; Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J.; Estrov, Zeev

    2014-01-01

    Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells. PMID:21364020

  1. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta

    2013-12-10

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568ASTAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. © Copyright 2014 Federation of European Biochemical Societies. All rights reserved.

  2. Correlation of methane production and functional gene transcriptional activity in a peat soil.

    Science.gov (United States)

    Freitag, Thomas E; Prosser, James I

    2009-11-01

    The transcription dynamics of subunit A of the key gene in methanogenesis (methyl coenzyme M reductase; mcrA) was studied to evaluate the relationship between process rate (methanogenesis) and gene transcription dynamics in a peat soil ecosystem. Soil methanogen process rates were determined during incubation of peat slurries at temperatures from 4 to 37 degrees C, and real-time quantitative PCR was applied to quantify the abundances of mcrA genes and transcripts; corresponding transcriptional dynamics were calculated from mcrA transcript/gene ratios. Internal standards suggested unbiased recovery of mRNA abundances in comparison to DNA levels. In comparison to those in pure-culture studies, mcrA transcript/gene ratios indicated underestimation by 1 order of magnitude, possibly due to high proportions of inactive or dead methanogens. Methane production rates were temperature dependent, with maxima at 25 degrees C, but changes in abundance and transcription of the mcrA gene showed no correlation with temperature. However, mcrA transcript/gene ratios correlated weakly (regression coefficient = 0.76) with rates of methanogenesis. Methanogen process rates increased over 3 orders of magnitude, while the corresponding maximum transcript/gene ratio increase was only 18-fold. mcrA transcript dynamics suggested steady-state expression in peat soil after incubation for 24 and 48 h, similar to that in stationary-phase cultures. mcrA transcript/gene ratios are therefore potential in situ indicators of methanogen process rate changes in complex soil systems.

  3. LIM homeobox transcription factor Lhx2 inhibits skeletal muscle differentiation in part via transcriptional activation of Msx1 and Msx2.

    Science.gov (United States)

    Kodaka, Yusaku; Tanaka, Kiyoko; Kitajima, Kenji; Tanegashima, Kosuke; Matsuda, Ryoichi; Hara, Takahiko

    2015-02-15

    LIM homeobox transcription factor Lhx2 is known to be an important regulator of neuronal development, homeostasis of hair follicle stem cells, and self-renewal of hematopoietic stem cells; however, its function in skeletal muscle development is poorly understood. In this study, we found that overexpression of Lhx2 completely inhibits the myotube-forming capacity of C2C12 cells and primary myoblasts. The muscle dedifferentiation factors Msx1 and Msx2 were strongly induced by the Lhx2 overexpression. Short interfering RNA-mediated knockdown of Lhx2 in the developing limb buds of mouse embryos resulted in a reduction in Msx1 and Msx2 mRNA levels, suggesting that they are downstream target genes of Lhx2. We found two Lhx2 consensus-binding sites in the -2097 to -1189 genomic region of Msx1 and two additional sites in the -536 to +73 genomic region of Msx2. These sequences were shown by luciferase reporter assay to be essential for Lhx2-mediated transcriptional activation. Moreover, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that Lhx2 is present in chromatin DNA complexes bound to the enhancer regions of the Msx1 and Msx2 genes. These data demonstrate that Msx1 and Msx2 are direct transcriptional targets of Lhx2. In addition, overexpression of Lhx2 significantly enhanced the mRNA levels of bone morphogenetic protein 4 and transforming growth factor beta family genes. We propose that Lhx2 is involved in the early stage of skeletal muscle development by inducing multiple differentiation inhibitory factors.

  4. Lycopene activates antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers.

    Science.gov (United States)

    Sahin, K; Orhan, C; Tuzcu, M; Sahin, N; Hayirli, A; Bilgili, S; Kucuk, O

    2016-05-01

    This study was conducted to evaluate the effects of dietary lycopene supplementation on growth performance, antioxidant status, and muscle nuclear transcription factor [Kelch like-ECH-associated protein 1 (Keap1) and (erythroid-derived 2)-like 2 (Nrf2)] expressions in broiler chickens exposed to heat stress (HS). A total of 180 one-day-old male broiler chicks (Ross 308) were assigned randomly to one of 2×3 factorially arranged treatments: two housing temperatures (22°C for 24 h/d; thermoneutral, TN or 34°C for 8 h/d HS) and three dietary lycopene levels (0, 200, or 400 mg/kg). Each treatment consisted of three replicates of 10 birds. Birds were reared to 42 d of age. Heat stress caused reductions in feed intake and weight gain by 12.2 and 20.7% and increased feed efficiency by 10.8% (Plycopene level improved performance in both environments. Birds reared under the HS environment had lower serum and muscle lycopene concentration (0.34 vs. 0.50 μg/mL and 2.80 vs. 2.13 μg/g), activities of superoxide dismutase (151 vs. 126 U/mL and 131 vs. 155 U/mg protein), glutathione peroxidase (184 vs. 154 U/mL and 1.39 vs. 1.74 U/mg protein), and higher malondialdehyde (MDA) concentration (0.53 vs. 0.83 μg/mL and 0.78 vs. 0.45 μg/ mg protein) than birds reared under the TN environment. Changes in levels of lycopene and MDA and activities of enzymes in serum and muscle varied by the environmental temperature as dietary lycopene level increased. Moreover, increasing dietary lycopene level suppressed muscle Keap1 expression and enhanced muscle Nrf2 expression, which had increased by 150% and decreased by 40%, respectively in response to HS. In conclusion, lycopene supplementation alleviates adverse effects of HS on performance through modulating expressions of stress-related nuclear transcription factors.

  5. ISOBUTYRAMIDE ACTIVATES TRANSCRIPTION OF HUMAN FETAL γ- AND MURINE EMBRYONIC εy-GLOBIN GENES

    Institute of Scientific and Technical Information of China (English)

    张俊武; 张雪青; 陈平

    2001-01-01

    Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globingene expression and to test cell toxicity of the drug.Methods. ME L cells were transfected with the recombinant construct μLCRAγψβδβ3 and the stable transfor-mants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rab-bit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection as-say. The hematologdcal toxicity and electrolyte toxicity of the drug were tested.Results. An inducible and dose-dependent expression of the human γγ-, ββ- and mouse Aa-globin gene was ob-served in the transfected MEL cells. The induction of the human γ-globin gene is significant stronger than that ofthe ββ-globin gene. With 2.5 ~ 5 mmol/L isobutyramide, the induction of the human γ-globin gene is even moreeffective than that of mouse aa-globin gene. After a 15-day injection under the doses of 500 ~ 900mg * kg- 1 * d-1,the level of the mouse embryonic εy-globin mRNA could be significantly induced up to 3 ~ 4 fold of that of uninject-ed controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected withdifferent doses of isobutyramide at the interval of 24 hours for 2 ~4 weeks, were generally within the normalrange. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+,Na +, C1- and CO.2 were all within normal range and serum ionic osmotic pressure remained stable as well.Conclusion. Our results suggested that isobutyramide is a weak inducer of cell differentiation, but it canselectively activate transcription of human γy-globin gene at a certain degree, and it can act on early stages of ery-throid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have nohematological toxicity. Our results have further proved the potential value of

  6. GCN5 is essential for IRF-4 gene expression followed by transcriptional activation of Blimp-1 in immature B cells.

    Science.gov (United States)

    Kikuchi, Hidehiko; Nakayama, Masami; Kuribayashi, Futoshi; Imajoh-Ohmi, Shinobu; Nishitoh, Hideki; Takami, Yasunari; Nakayama, Tatsuo

    2014-03-01

    During B-cell differentiation, the gene expression of B-cell differentiation-related transcription factors must be strictly controlled by epigenetic mechanisms including histone acetylation and deacetylation, to complete the differentiation pathway. GCN5, one of the most important histone acetyltransferases, is involved in epigenetic events for transcriptional regulation through alterations in the chromatin structure. In this study, by analyzing the homozygous DT40 mutants GCN5(-/-), generated with gene targeting techniques, we found that GCN5 was necessary for transcriptional activation of IRF-4, an essential transcription factor for plasma cell differentiation. GCN5 deficiency caused drastic decreases in both the mRNA and the protein levels of Blimp-1 and IRF-4. The ectopic expression of Blimp-1 and IRF-4 suggests that IRF-4, but not Blimp-1, is the target gene of GCN5 in immature B cells. Moreover, a chromatin immunoprecipitation assay showed that GCN5 bound to the IRF-4 gene around its 5'-flanking region and acetylated H3K9 residues within chromatin surrounding the region in vivo, suggesting that gene expression of IRF-4 is certainly regulated by GCN5. These results reveal that GCN5 is essential for IRF-4 gene expression, followed by transcriptional activation of Blimp-1, and plays a key role in epigenetic regulation of B-cell differentiation.

  7. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    Energy Technology Data Exchange (ETDEWEB)

    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan); Oritani, Kenji [Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Matsuda, Tadashi, E-mail: tmatsuda@pharm.hokudai.ac.jp [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan)

    2015-07-31

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR.

  8. Involvement of multiple elements in FXR-mediated transcriptional activation of FGF19.

    Science.gov (United States)

    Miyata, Masaaki; Hata, Tatsuya; Yamakawa, Hiroki; Kagawa, Tatehiro; Yoshinari, Kouichi; Yamazoe, Yasushi

    2012-10-01

    The intestinal endocrine hormone human fibroblast growth factor 19 (FGF19) is involved in the regulation of not only hepatic bile acid metabolism but also carbohydrate and lipid metabolism. In the present study, bile acid/farnesoid X receptor (FXR) responsiveness in the FGF19 promoter region was investigated by a reporter assay using the human colon carcinoma cell line LS174T. The assay revealed the presence of bile acid/FXR-responsive elements in the 5'-flanking region up to 8.8 kb of FGF19. Deletion analysis indicated that regions from -1866 to -1833, from -1427 to -1353, and from -75 to +262 were involved in FXR responsiveness. Four, four, and two consecutive half-sites of nuclear receptors were observed in the three regions, respectively. An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay revealed FXR/retinoid X receptor α (RXRα) heterodimer binding in these three regions. EMSA and reporter assays using mutated constructs indicated that the nuclear receptor IR1, ER2, and DR8 motifs in the 5'-flanking region were involved in FXR responsiveness of FGF19. Lithocholic acid (LCA) (10 μM), chenodeoxycholic acid (CDCA) (10 μM), or GW4064 (0.1 μM) treatment increased reporter activity in a construct including the three motifs under FXR-expressing conditions whereas LCA and not CDCA or GW4064 treatment increased the reporter activity under pregnane X receptor (PXR)-expressing conditions. These results suggest that FGF19 is transcriptionally activated through multiple FXR-responsive elements in the promoter region.

  9. Epidermal growth factor receptor regulates β-catenin location, stability, and transcriptional activity in oral cancer

    Directory of Open Access Journals (Sweden)

    Hung Hsing-Wen

    2010-03-01

    Full Text Available Abstract Background Many cancerous cells accumulate β-catenin in the nucleus. We examined the role of epidermal growth factor receptor (EGFR signaling in the accumulation of β-catenin in the nuclei of oral cancer cells. Results We used two strains of cultured oral cancer cells, one with reduced EGFR expression (OECM1 cells and one with elevated EGFR expression (SAS cells, and measured downstream effects, such as phosphorylation of β-catenin and GSK-3β, association of β-catenin with E-cadherin, and target gene regulation. We also studied the expression of EGFR, β-catenin, and cyclin D1 in 112 samples of oral cancer by immunostaining. Activation of EGFR signaling increased the amount of β-catenin in the nucleus and decreased the amount in the membranes. EGF treatment increased phosphorylation of β-catenin (tyrosine and GSK-3β(Ser-(9, resulting in a loss of β-catenin association with E-cadherin. TOP-FLASH and FOP-FLASH reporter assays demonstrated that the EGFR signal regulates β-catenin transcriptional activity and mediates cyclin D1 expression. Chromatin immunoprecipitation experiments indicated that the EGFR signal affects chromatin architecture at the regulatory element of cyclin D1, and that the CBP, HDAC1, and Suv39h1 histone/chromatin remodeling complex is involved in this process. Immunostaining showed a significant association between EGFR expression and aberrant accumulation of β-catenin in oral cancer. Conclusions EGFR signaling regulates β-catenin localization and stability, target gene expression, and tumor progression in oral cancer. Moreover, our data suggest that aberrant accumulation of β-catenin under EGFR activation is a malignancy marker of oral cancer.

  10. Effects of chronic renal failure rat serum on histone acetyltransferase p300 and activation of activating transcription factor 4 of arterial smooth muscle cells cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    张耀全

    2014-01-01

    Objective To investigate the effects of the rat serum with chronic renal failure(CRF)on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4)of rat arterial vascular smooth muscle cells(VSMCs)cultured in vitro,and explore the possible mechanism.Methods Objective To establish the rat model of

  11. Transcriptional Activation of Low-Density Lipoprotein Receptor Gene by DJ-1 and Effect of DJ-1 on Cholesterol Homeostasis

    Science.gov (United States)

    Takahashi-Niki, Kazuko; Kato, Izumi; Niki, Takeshi; Goldberg, Matthew S.; Shen, Jie; Ishimoto, Kenji; Doi, Takefumi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2012-01-01

    DJ-1 is a novel oncogene and also causative gene for familial Parkinson’s disease park7. DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. For transcriptional regulation, DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found the low-density lipoprotein receptor (LDLR) gene is a transcriptional target gene for DJ-1. Reduced expression of LDLR mRNA and protein was observed in DJ-1-knockdown cells and DJ-1-knockout mice and this occurred at the transcription level. Reporter gene assays using various deletion and point mutations of the LDLR promoter showed that DJ-1 stimulated promoter activity by binding to the sterol regulatory element (SRE) with sterol regulatory element binding protein (SREBP) and that stimulating activity of DJ-1 toward LDLR promoter activity was enhanced by oxidation of DJ-1. Chromatin immunoprecipitation, gel-mobility shift and co-immunoprecipitation assays showed that DJ-1 made a complex with SREBP on the SRE. Furthermore, it was found that serum LDL cholesterol level was increased in DJ-1-knockout male, but not female, mice and that the increased serum LDL cholesterol level in DJ-1-knockout male mice was cancelled by administration with estrogen, suggesting that estrogen compensates the increased level of serum LDL cholesterol in DJ-1-knockout female mice. This is the first report that DJ-1 participates in metabolism of fatty acid synthesis through transcriptional regulation of the LDLR gene. PMID:22666465

  12. Transcriptional activation of low-density lipoprotein receptor gene by DJ-1 and effect of DJ-1 on cholesterol homeostasis.

    Directory of Open Access Journals (Sweden)

    Shiori Yamaguchi

    Full Text Available DJ-1 is a novel oncogene and also causative gene for familial Parkinson's disease park7. DJ-1 has multiple functions that include transcriptional regulation, anti-oxidative reaction and chaperone and mitochondrial regulation. For transcriptional regulation, DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found the low-density lipoprotein receptor (LDLR gene is a transcriptional target gene for DJ-1. Reduced expression of LDLR mRNA and protein was observed in DJ-1-knockdown cells and DJ-1-knockout mice and this occurred at the transcription level. Reporter gene assays using various deletion and point mutations of the LDLR promoter showed that DJ-1 stimulated promoter activity by binding to the sterol regulatory element (SRE with sterol regulatory element binding protein (SREBP and that stimulating activity of DJ-1 toward LDLR promoter activity was enhanced by oxidation of DJ-1. Chromatin immunoprecipitation, gel-mobility shift and co-immunoprecipitation assays showed that DJ-1 made a complex with SREBP on the SRE. Furthermore, it was found that serum LDL cholesterol level was increased in DJ-1-knockout male, but not female, mice and that the increased serum LDL cholesterol level in DJ-1-knockout male mice was cancelled by administration with estrogen, suggesting that estrogen compensates the increased level of serum LDL cholesterol in DJ-1-knockout female mice. This is the first report that DJ-1 participates in metabolism of fatty acid synthesis through transcriptional regulation of the LDLR gene.

  13. Rh D blood group conversion using transcription activator-like effector nucleases

    Science.gov (United States)

    Kim, Young-Hoon; Kim, Hyun O.; Baek, Eun J.; Kurita, Ryo; Cha, Hyuk-Jin; Nakamura, Yukio; Kim, Hyongbum

    2015-01-01

    Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine. PMID:26078220

  14. The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation.

    Science.gov (United States)

    Moskot, Marta; Montefusco, Sandro; Jakóbkiewicz-Banecka, Joanna; Mozolewski, Paweł; Węgrzyn, Alicja; Di Bernardo, Diego; Węgrzyn, Grzegorz; Medina, Diego L; Ballabio, Andrea; Gabig-Cimińska, Magdalena

    2014-06-13

    Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein-mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases.

  15. RAM-induced Allostery Facilitates Assembly of a Notch Pathway Active Transcription Complex

    Energy Technology Data Exchange (ETDEWEB)

    Friedmann, David R.; Wilson, Jeffrey J.; Kovall, Rhett A. (UCIN-MED)

    2008-07-09

    The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSL-NotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro.

  16. RAM-Induced Allostery Facilitates Assembly of a Notch Pathway Active Transcription Complex

    Energy Technology Data Exchange (ETDEWEB)

    Friedman,D.; Wilson, J.; Kovall, R.

    2008-01-01

    The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSL-NotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro.

  17. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

    KAUST Repository

    Lescot, Magali

    2015-11-27

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  18. hSSB1 regulates both the stability and the transcriptional activity of p53

    Institute of Scientific and Technical Information of China (English)

    Shuangbing Xu; Yuanzhong Wu; Qiong Chen; Jingying Cao; Kaishun Hu; Jianjun Tang; Yi Sang

    2013-01-01

    The tumor suppressor p53 is essential for several cellular processes that are involved in the response to diverse genotoxic stress,including cell cycle arrest,DNA repair,apoptosis and senescence.Studies of the regulation of p53 have mostly focused on its stability and transactivation; however,new regulatory molecules for p53 have also been frequently identified.Here,we report that human ssDNA binding protein SSB1 (hSSB1),a novel DNA damageassociated protein,can interact with p53 and protect p53 from ubiquitin-mediated degradation.Furthermore,hSSB1 also associates with the acetyltransferase p300 and is required for efficient transcriptional activation of the p53 target gene p21 by affecting the acetylation of p53 at lysine382.Functionally,the hSSB1 knockdown-induced abrogation of the G2/M checkpoint is partially dependent on p53 or p300.Collectively,our results indicate that hSSB1 may regulate DNA damage checkpoints by positively modulating p53 and its downstream target p21.

  19. Genome-wide analysis of LXRα activation reveals new transcriptional networks in human atherosclerotic foam cells.

    Science.gov (United States)

    Feldmann, Radmila; Fischer, Cornelius; Kodelja, Vitam; Behrens, Sarah; Haas, Stefan; Vingron, Martin; Timmermann, Bernd; Geikowski, Anne; Sauer, Sascha

    2013-04-01

    Increased physiological levels of oxysterols are major risk factors for developing atherosclerosis and cardiovascular disease. Lipid-loaded macrophages, termed foam cells, are important during the early development of atherosclerotic plaques. To pursue the hypothesis that ligand-based modulation of the nuclear receptor LXRα is crucial for cell homeostasis during atherosclerotic processes, we analysed genome-wide the action of LXRα in foam cells and macrophages. By integrating chromatin immunoprecipitation-sequencing (ChIP-seq) and gene expression profile analyses, we generated a highly stringent set of 186 LXRα target genes. Treatment with the nanomolar-binding ligand T0901317 and subsequent auto-regulatory LXRα activation resulted in sequence-dependent sharpening of the genome-binding patterns of LXRα. LXRα-binding loci that correlated with differential gene expression revealed 32 novel target genes with potential beneficial effects, which in part explained the implications of disease-associated genetic variation data. These observations identified highly integrated LXRα ligand-dependent transcriptional networks, including the APOE/C1/C4/C2-gene cluster, which contribute to the reversal of cholesterol efflux and the dampening of inflammation processes in foam cells to prevent atherogenesis.

  20. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages.

    Science.gov (United States)

    Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Iudicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

    2016-05-01

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  1. Crystal structure of the caseinolytic protease gene regulator, a transcriptional activator in actinomycetes.

    Science.gov (United States)

    Russo, Santina; Schweitzer, Jens-Eric; Polen, Tino; Bott, Michael; Pohl, Ehmke

    2009-02-20

    Human pathogens of the genera Corynebacterium and Mycobacterium possess the transcriptional activator ClgR (clp gene regulator) which in Corynebacterium glutamicum has been shown to regulate the expression of the ClpCP protease genes. ClgR specifically binds to pseudo-palindromic operator regions upstream of clpC and clpP1P2. Here, we present the first crystal structure of a ClgR protein from C. glutamicum. The structure was determined from two different crystal forms to resolutions of 1.75 and 2.05 A, respectively. ClgR folds into a five-helix bundle with a helix-turn-helix motif typical for DNA-binding proteins. Upon dimerization the two DNA-recognition helices are arranged opposite to each other at the protein surface in a distance of approximately 30 A, which suggests that they bind into two adjacent major grooves of B-DNA in an anti-parallel manner. A binding pocket is situated at a strategic position in the dimer interface and could possess a regulatory role altering the positions of the DNA-binding helices.

  2. The ETS-5 transcription factor regulates activity states in Caenorhabditis elegans by controlling satiety

    Science.gov (United States)

    Juozaityte, Vaida; Pladevall-Morera, David; Podolska, Agnieszka; Nørgaard, Steffen; Pocock, Roger

    2017-01-01

    Animal behavior is shaped through interplay among genes, the environment, and previous experience. As in mammals, satiety signals induce quiescence in Caenorhabditis elegans. Here we report that the C. elegans transcription factor ETS-5, an ortholog of mammalian FEV/Pet1, controls satiety-induced quiescence. Nutritional status has a major influence on C. elegans behavior. When foraging, food availability controls behavioral state switching between active (roaming) and sedentary (dwelling) states; however, when provided with high-quality food, C. elegans become sated and enter quiescence. We show that ETS-5 acts to promote roaming and inhibit quiescence by setting the internal “satiety quotient” through fat regulation. Acting from the ASG and BAG sensory neurons, we show that ETS-5 functions in a complex network with serotonergic and neuropeptide signaling pathways to control food-regulated behavioral state switching. Taken together, our results identify a neuronal mechanism for controlling intestinal fat stores and organismal behavioral states in C. elegans, and establish a paradigm for the elucidation of obesity-relevant mechanisms. PMID:28193866

  3. Generation of myostatin B knockout yellow catfish (Tachysurus fulvidraco) using transcription activator-like effector nucleases.

    Science.gov (United States)

    Dong, Zhangji; Ge, Jiachun; Xu, Zhiqiang; Dong, Xiaohua; Cao, Shasha; Pan, Jianlin; Zhao, Qingshun

    2014-06-01

    Myostatin (Mstn), a member of the transforming growth factor β superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnb(nju22), an 8 bp deletion, and mstnb(nju24), a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth.

  4. Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs).

    Science.gov (United States)

    Kowalko, Johanna E; Ma, Li; Jeffery, William R

    2016-06-20

    Identifying alleles of genes underlying evolutionary change is essential to understanding how and why evolution occurs. Towards this end, much recent work has focused on identifying candidate genes for the evolution of traits in a variety of species. However, until recently it has been challenging to functionally validate interesting candidate genes. Recently developed tools for genetic engineering make it possible to manipulate specific genes in a wide range of organisms. Application of this technology in evolutionarily relevant organisms will allow for unprecedented insight into the role of candidate genes in evolution. Astyanax mexicanus (A. mexicanus) is a species of fish with both surface-dwelling and cave-dwelling forms. Multiple independent lines of cave-dwelling forms have evolved from ancestral surface fish, which are interfertile with one another and with surface fish, allowing elucidation of the genetic basis of cave traits. A. mexicanus has been used for a number of evolutionary studies, including linkage analysis to identify candidate genes responsible for a number of traits. Thus, A. mexicanus is an ideal system for the application of genome editing to test the role of candidate genes. Here we report a method for using transcription activator-like effector nucleases (TALENs) to mutate genes in surface A. mexicanus. Genome editing using TALENs in A. mexicanus has been utilized to generate mutations in pigmentation genes. This technique can also be utilized to evaluate the role of candidate genes for a number of other traits that have evolved in cave forms of A. mexicanus.

  5. Rh D blood group conversion using transcription activator-like effector nucleases.

    Science.gov (United States)

    Kim, Young-Hoon; Kim, Hyun O; Baek, Eun J; Kurita, Ryo; Cha, Hyuk-Jin; Nakamura, Yukio; Kim, Hyongbum

    2015-06-16

    Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

  6. The role of signal transducer and activator of transcription 3 in Rift Valley fever virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Pinkham, Chelsea; An, Soyeon; Lundberg, Lindsay; Bansal, Neha; Benedict, Ashwini; Narayanan, Aarthi; Kehn-Hall, Kylene, E-mail: kkehnhal@gmu.edu

    2016-09-15

    Rift Valley fever (RVF) is a zoonotic disease that can cause severe illness in humans and livestock, triggering spontaneous abortion in almost 100% of pregnant ruminants. In this study, we demonstrate that signal transducer and activator of transcription 3 (STAT3) is phosphorylated on its conserved tyrosine residue (Y705) following RVFV infection. This phosphorylation was dependent on a major virulence factor, the viral nonstructural protein NSs. Loss of STAT3 had little effect on viral replication, but rather resulted in cells being more susceptible to RVFV-induced cell death. Phosphorylated STAT3 translocated to the nucleus, coinciding with inhibition of fos, jun, and nr4a2 gene expression, and the presence of STAT3 and NSs at the nr4a2 promoter. NSs was found predominantly in the cytoplasm of STAT3 null cells, indicating that STAT3 influences NSs nuclear localization. Collectively, these data demonstrate that STAT3 functions in a pro-survival capacity through modulation of NSs localization. - Highlights: • STAT3 is phosphorylated on tyrosine residue 705 following RVFV infection. • Phosphorylation of STAT3 was dependent on the viral protein NSs. • STAT3 -/- MEFs were more susceptible to RVFV-induced cell death. • Loss of STAT3 led to an increase in pro-apoptotic gene expression. • STAT3 functions in a pro-survival capacity by modulation of NSs localization.

  7. Critical Role of Activating Transcription Factor 4 in the Anabolic Actions of Parathyroid Hormone in Bone

    Science.gov (United States)

    Yu, Shibing; Franceschi, Renny T.; Luo, Min; Fan, Jie; Jiang, Di; Cao, Huiling; Kwon, Tae-Geon; Lai, Yumei; Zhang, Jian; Patrene, Kenneth; Hankenson, Kurt; Roodman, G. David; Xiao, Guozhi

    2009-01-01

    Parathyroid hormone (PTH) is a potent anabolic agent for the treatment of osteoporosis. However, its mechanism of action in osteoblast and bone is not well understood. In this study, we show that the anabolic actions of PTH in bone are severely impaired in both growing and adult ovariectomized mice lacking bone-related activating transcription factor 4 (ATF4). Our study demonstrates that ATF4 deficiency suppresses PTH-stimulated osteoblast proliferation and survival and abolishes PTH-induced osteoblast differentiation, which, together, compromise the anabolic response. We further demonstrate that the PTH-dependent increase in osteoblast differentiation is correlated with ATF4-dependent up-regulation of Osterix. This regulation involves interactions of ATF4 with a specific enhancer sequence in the Osterix promoter. Furthermore, actions of PTH on Osterix require this same element and are associated with increased binding of ATF4 to chromatin. Taken together these experiments establish a fundamental role for ATF4 in the anabolic actions of PTH on the skeleton. PMID:19851510

  8. Critical role of activating transcription factor 4 in the anabolic actions of parathyroid hormone in bone.

    Directory of Open Access Journals (Sweden)

    Shibing Yu

    Full Text Available Parathyroid hormone (PTH is a potent anabolic agent for the treatment of osteoporosis. However, its mechanism of action in osteoblast and bone is not well understood. In this study, we show that the anabolic actions of PTH in bone are severely impaired in both growing and adult ovariectomized mice lacking bone-related activating transcription factor 4 (ATF4. Our study demonstrates that ATF4 deficiency suppresses PTH-stimulated osteoblast proliferation and survival and abolishes PTH-induced osteoblast differentiation, which, together, compromise the anabolic response. We further demonstrate that the PTH-dependent increase in osteoblast differentiation is correlated with ATF4-dependent up-regulation of Osterix. This regulation involves interactions of ATF4 with a specific enhancer sequence in the Osterix promoter. Furthermore, actions of PTH on Osterix require this same element and are associated with increased binding of ATF4 to chromatin. Taken together these experiments establish a fundamental role for ATF4 in the anabolic actions of PTH on the skeleton.

  9. Direct observation of transcription activator-like effector (TALE) protein dynamics

    Science.gov (United States)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M.

    2014-03-01

    In this work, we describe a single molecule assay to probe the site-search dynamics of transcription activator-like effector (TALE) proteins along DNA. In modern genetics, the ability to selectively edit the human genome is an unprecedented development, driven by recent advances in targeted nuclease proteins. Specific gene editing can be accomplished using TALE proteins, which are programmable DNA-binding proteins that can be fused to a nuclease domain. In this way, TALENs are a leading technology that has shown great success in the genomic editing of pluripotent stem cells. A major hurdle facing clinical implementation, however, is the potential for deleterious off-target binding events. For these reasons, a molecular-level understanding of TALE binding and target sequence search on DNA is essential. To this end, we developed a single-molecule fluorescence imaging assay that provides a first-of-its-kind view of the 1-D diffusion of TALE proteins along stretched DNA. Taken together with co-crystal structures of DNA-bound TALEs, our results suggest a rotationally-coupled, major groove tracking model for diffusion. We further report diffusion constants for TALE proteins as a function of salt concentration, consistent with previously described models of 1-D protein diffusion.

  10. EBV tegument protein BNRF1 disrupts DAXX-ATRX to activate viral early gene transcription.

    Directory of Open Access Journals (Sweden)

    Kevin Tsai

    2011-11-01

    Full Text Available Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs, suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus.

  11. Identification of retrotransposon families and analysis of their transcriptional activation in wheat

    Institute of Scientific and Technical Information of China (English)

    TANG Yimiao; MA Youzhi; LI Liancheng; XIN Zhiyong

    2005-01-01

    Variations in the reverse transcriptase (RT) domain were analyzed to clarify retrotransposon family structure and their evolution in wheat genome. RT-PCR was conducted by using primers based on the conserved RT peptide motifs of plant retrotransposons to amplify the RT domain of retrotransposons in the seedlings of wheat line Pm97034 treated with powdery mildew fungus. High level of heterogeneity was detected in 51 (RT1-51) clones randomly selected and the identity of nucleotide sequence among them ranged from 75.4% to 97.9%. These sequences, in combination with previously identified seven representatives from wheat retrotransposon families (families 1―7), were used to construct a composite phylogenetic tree. Three new families, designated family 8 to family 10, were identified. Famliy 8 formed before divergence of the Pooideae subfamily and was regarged as an ancient retrotransposon family. Some members of family 4 and family 7 had transcriptional activativity, and were present with multiple copies in wheat and its related species.

  12. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular tran-scriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells’ entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  13. Immediate-early gene product ICP22 inhibits the trans-transcription activating function of P53-mdm-2

    Institute of Scientific and Technical Information of China (English)

    GUO HongXiong; CUN Wei; LIU LongDing; WANG LiChun; ZHAO HongLing; DONG ChengHong; LI QiHan

    2007-01-01

    As a product of HSVI immediate-early gene, ICP22 is capable of interacting with various cellular transcriptive and regulatory molecules during viral infection so as to impact the normal cellular molecular mechanism. ICP22 expressed in transfected cells can push the cells' entering into S phase with binding to mdm-1 promoter region and impact its trans-transcription activating effect by P53. Consequently, the MDM-2 binds to P53, and the degradation effects by the ubiquitous pathway are decreased, improving indirectly the P53 levels in cells and making the cells progress into the S phase.

  14. Transcriptional profiling of the dose response: a more powerful approach for characterizing drug activities.

    Directory of Open Access Journals (Sweden)

    Rui-Ru Ji

    2009-09-01

    Full Text Available The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901 across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

  15. Histone deacetylases regulate gonadotropin-releasing hormone I gene expression via modulating Otx2-driven transcriptional activity.

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    Lu Gan

    Full Text Available BACKGROUND: Precise coordination of the hypothalamic-pituitary-gonadal axis orchestrates the normal reproductive function. As a central regulator, the appropriate synthesis and secretion of gonadotropin-releasing hormone I (GnRH-I from the hypothalamus is essential for the coordination. Recently, emerging evidence indicates that histone deacetylases (HDACs play an important role in maintaining normal reproductive function. In this study, we identify the potential effects of HDACs on Gnrh1 gene transcription. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of HDACs activities by trichostatin A (TSA and valproic acid (VPA promptly and dramatically repressed transcription of Gnrh1 gene in the mouse immortalized mature GnRH neuronal cells GT1-7. The suppression was connected with a specific region of Gnrh1 gene promoter, which contains two consensus Otx2 binding sites. Otx2 has been known to activate the basal and also enhancer-driven transcription of Gnrh1 gene. The transcriptional activity of Otx2 is negatively modulated by Grg4, a member of the Groucho-related-gene (Grg family. In the present study, the expression of Otx2 was downregulated by TSA and VPA in GT1-7 cells, accompanied with the opposite changes of Grg4 expression. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the DNA-binding activity of Otx2 to Gnrh1 gene was suppressed by TSA and VPA. Overexpression of Otx2 partly abolished the TSA- and VPA-induced downregulation of Gnrh1 gene expression. CONCLUSIONS/SIGNIFICANCE: Our data indicate that HDAC inhibitors downregulate Gnrh1 gene expression via repressing Otx2-driven transcriptional activity. This study should provide an insight for our understanding on the effects of HDACs in the reproductive system and suggests that HDACs could be potential novel targets for the therapy of GnRH-related diseases.

  16. Activation of bone morphogenetic protein-6 gene transcription in MCF-7 cells by estrogen

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ming; YAN Ji-dong; HANG Lei; WANG Qing; L(U) Shu-jun; ZHANG Jie; ZHU Tian-hui

    2005-01-01

    Background Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17β-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER+) breast cancer cell line MCF-7. Methods After the treatment of MCF-7 cells with E2 at different concentrations (10-11 mol/L, 10-9 mol/L, 10-7 mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0.7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10-7 mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half-site (1/2 ERE) element and Sp1 sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (ChIP) assay was also used to confirm the binding of estrogen receptor α (Erα) on BMP-6 promoter in the presence of E2. Results E2 dose dependently increased BMP-6 mRNA expression in human ER+ breast cancer cell line MCF-7. At a dose of 10-7 mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P<0.05). Both the 1/2 ERE response element mutant form and the Sp1 site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter's response to E2. Through ChIP assay, the binding of Erα on 1/2 ERE response element in BMP-6 promoter was further validated. Conclusion Estrogen induces BMP-6 expression in human ER+ breast cancer cell line MCF-7 through its receptor Erα binding on 1

  17. The emerging regulatory potential of SCFMet30 -mediated polyubiquitination and proteolysis of the Met4 transcriptional activator

    Directory of Open Access Journals (Sweden)

    Chandrasekaran Srikripa

    2008-07-01

    Full Text Available Abstract The yeast SCFMet30 ubiquitin ligase plays a critical role in cell division by regulating the Met4 transcriptional activator of genes that control the uptake and assimilation of sulfur into methionine and S-adenosyl-methionine. The initial view on how SCFMet30 performs its function has been driven by the assumption that SCFMet30 acts exclusively as Met4 inhibitor when high levels of methionine drive an accumulation of cysteine. We revisit this model in light of the growing evidence that SCFMet30 can also activate Met4. The notion that Met4 can be inhibited or activated depending on the sulfur metabolite context is not new, but for the first time both aspects have been linked to SCFMet30, creating an interesting regulatory paradigm in which polyubiquitination and proteolysis of a single transcriptional activator can play different roles depending on context. We discuss the emerging molecular basis and the implications of this new regulatory phenomenon.

  18. Regulating retrotransposon activity through the use of alternative transcription start sites

    DEFF Research Database (Denmark)

    Persson, Jenna; Steglich, Babett; Smialowska, Agata;

    2016-01-01

    a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements....... This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled...

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

    OpenAIRE

    2010-01-01

    Abstract Frequent mutations of p53 in human cancers exemplify its crucial role as a tumor suppressor transcription factor, and p21, a transcriptional target of p53, plays a central role in surveillance of cell-cycle checkpoints. Our previous study has shown that FATS stabilize p21 to preserve genome integrity. In this study we identified a novel transcript variant of FATS (GenBank: GQ499374) through screening a cDNA library from mouse testis, which uncovered the promoter region of mouse FATS....

  20. The loss of activating transcription factor 4 (ATF4) reduces bone toughness and fracture toughness.

    Science.gov (United States)

    Makowski, Alexander J; Uppuganti, Sasidhar; Wadeer, Sandra A; Whitehead, Jack M; Rowland, Barbara J; Granke, Mathilde; Mahadevan-Jansen, Anita; Yang, Xiangli; Nyman, Jeffry S

    2014-05-01

    Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of these important factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4-/- littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4-/- mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective of age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4-/- mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also in maintaining bone toughness and fracture toughness.

  1. UNTAGGED MUTATION IN RICE GAL4/VP16 TRANSCRIPTIONAL ACTIVATOR FACILITATED-ENHANCER TRAP LINES

    Directory of Open Access Journals (Sweden)

    Sri Koerniati

    2013-04-01

    Full Text Available An enhancer trap system is an insertional mutagenesis based upon gene expression, instead of gene knock-out, so its insertion in genome is  expected not linked to any dramatic changes in plant phenotypes. Gene  knock-out, leading to lossof- function (LoF mutation, is a dominant  approach for rice functional genomic studies. The objective of this study was to find out whether Transcriptional Activator-Facilitated Enhancer Trap (TAFET T-DNA insertion inducing mutant phenotypes in rice TAFET population. Materials used in this experiment were T1 generation of 270 rice TAFET lines. Eight plants of each were grown in the greenhouse and observed for any mutant phenotypes. Phenotypic, histochemical, Southernblot analyses were carried out to define a mutant of pSKC66.1- 8e. Result showed that about 10% of the 270 lines produced chlorophyll-deficient  leaves, ranged from yellowish green (viridis, white stripe green zebra-like stripe to completely white (albino. Albino plants died after two weeks,  whilst white stripe or viridis mutants became normal in the next generation(T2. Another mutant was pSKC66.1-8e line which had floral dramatic phenotype change with various spikelet shapes and number of organs, and had a single twisted culm. The flower of mutant also had gus gene expression. Plants with wild type did not express gus gene and had six or more straight culms. Molecular, histochemical and phenotypic analyses of this particular line for three generations indicated that mutant phenotype was not due to the T-DNA insertion. Since there was approved that Tos17 is activated during tissue culture and induced mutant phenotype, this line might relate to Tos17 insertion, but it needs further investigation to gain such conclusion.

  2. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

    Directory of Open Access Journals (Sweden)

    Finola E Moore

    Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  3. The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

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    Francesca Carlini

    Full Text Available BACKGROUND: Transposable Elements (TEs comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1 and Human Endogenous Retroviruses (HERVs that code for their own endogenous reverse transcriptase (RT. Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC, a nucleoside reverse transcription inhibitor (NRTI, on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

  4. Functional diversification of FD transcription factors in rice, components of florigen activation complexes.

    Science.gov (United States)

    Tsuji, Hiroyuki; Nakamura, Hiroyuki; Taoka, Ken-ichiro; Shimamoto, Ko

    2013-03-01

    Florigen, a protein encoded by the FLOWERING LOCUS T (FT) in Arabidopsis and Heading date 3a (Hd3a) in rice, is the universal flowering hormone in plants. Florigen is transported from leaves to the shoot apical meristem and initiates floral evocation. In shoot apical cells, conserved cytoplasmic 14-3-3 proteins act as florigen receptors. A hexameric florigen activation complex (FAC) composed of Hd3a, 14-3-3 proteins, and OsFD1, a transcription factor, activates OsMADS15, a rice homolog of Arabidopsis APETALA1, leading to flowering. Because FD is a key component of the FAC, we characterized the FD gene family and their functions. Phylogenetic analysis of FD genes indicated that this family is divided into two groups: (i) canonical FD genes that are conserved among eudicots and non-Poaceae monocots; and (ii) Poaceae-specific FD genes that are organized into three subgroups: Poaceae FD1, FD2 and FD3. The Poaceae FD1 group shares a small sequence motif, T(A/V)LSLNS, with FDs of eudicots and non-Poaceae monocots. Overexpression of OsFD2, a member of the Poaceae FD2 group, produced smaller leaves with shorter plastochrons, suggesting that OsFD2 controls leaf development. In vivo subcellular localization of Hd3a, 14-3-3 and OsFD2 suggested that in contrast to OsFD1, OsFD2 is restricted to the cytoplasm through its interaction with the cytoplasmic 14-3-3 proteins, and interaction of Hd3a with 14-3-3 facilitates nuclear translocation of the FAC containing OsFD2. These results suggest that FD function has diverged between OsFD1 and OsFD2, but formation of a FAC is essential for their function.

  5. The Proteasome Activator PA28γ, a Negative Regulator of p53, Is Transcriptionally Up-Regulated by p53

    Directory of Open Access Journals (Sweden)

    Zhen-Xing Wan

    2014-02-01

    Full Text Available PA28γ (also called REGγ, 11Sγ or PSME3 negatively regulates p53 activity by promoting its nuclear export and/or degradation. Here, using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE method, we identified the transcription start site of the PA28γ gene. Assessment with the luciferase assay demonstrated that the sequence −193 to +16 is the basal promoter. Three p53 binding sites were found within the PA28γ promoter utilizing a bioinformatics approach and were confirmed by chromatin immunoprecipitation and biotinylated DNA affinity precipitation experiments. The p53 protein promotes PA28γ transcription, and p53-stimulated transcription of PA28γ can be inhibited by PA28γ itself. Our results suggest that PA28γ and p53 form a negative feedback loop, which maintains the balance of p53 and PA28γ in cells.

  6. Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice

    OpenAIRE

    Hardmeier, Rosmarie; Hoeger, Harald; Fang-Kircher, Susanne; Khoschsorur, Ali; Lubec, Gert

    1997-01-01

    The involvement of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in radiobiological processes has been described at the enzyme activity level. We irradiated radiation-resistant (RR) and radiation-sensitive (RS) mice and studied antioxidant enzymes at the transcriptional and activity level. In addition, aromatic hydroxylation and lipid peroxidation parameters were determined to study radiation resistance at the oxidation level. RS BALB/c/J Him m...

  7. Simultaneous Gene Editing by Injection of mRNAs Encoding Transcription Activator-Like Effector Nucleases into Mouse Zygotes

    OpenAIRE

    Li, Chunliang; Qi, Rong; Singleterry, Rebecca; Hyle, Judith; Balch, Amanda; Li, Xiuling; Sublett, Jack; Berns, Hartmut; Valentine, Marcus; Valentine, Virginia; Sherr, Charles J.

    2014-01-01

    Injection of transcription activator-like effector nucleases (TALEN) mRNAs into mouse zygotes transferred into foster mothers efficiently generated founder mice with heritable mutations in targeted genes. Immunofluorescence visualization of phosphorylated histone 2A (γH2AX) combined with fluorescence in situ hybridization revealed that TALEN pairs targeting the Agouti locus induced site-directed DNA breaks in zygotes within 6 h of injection, an activity that continued at reduced efficiency in...

  8. Mécanismes de l'activation de la transcription in vivo par le Médiateur

    OpenAIRE

    Eyboulet, Fanny

    2014-01-01

    In eukaryotes, the synthesis of messenger RNA (mRNA) is highly regulated in response to the binding of specific activators to regulatory regions. This step allows the recruitment of coactivators, general transcription factors (GTFs) and RNA polymerase II (Pol II) to form the preinitiation complex (PIC). Mediator is a co-activator complex essential to this process and although it has been studied intensively during the last few years, its complexity has precluded a detailed understanding of th...

  9. GCN-2 dependent inhibition of protein synthesis activates osmosensitive gene transcription via WNK and Ste20 kinase signaling.

    Science.gov (United States)

    Lee, Elaine Choung-Hee; Strange, Kevin

    2012-12-15

    Increased gpdh-1 transcription is required for accumulation of the organic osmolyte glycerol and survival of Caenorhabditis elegans during hypertonic stress. Our previous work has shown that regulators of gpdh-1 (rgpd) gene knockdown constitutively activates gpdh-1 expression. Fifty-five rgpd genes play essential roles in translation suggesting that inhibition of protein synthesis is an important signal for regulating osmoprotective gene transcription. We demonstrate here that translation is reduced dramatically by hypertonic stress or knockdown of rgpd genes encoding aminoacyl-tRNA synthetases and eukaryotic translation initiation factors (eIFs). Toxin-induced inhibition of translation also activates gpdh-1 expression. Hypertonicity-induced translation inhibition is mediated by general control nonderepressible (GCN)-2 kinase signaling and eIF-2α phosphoryation. Loss of gcn-1 or gcn-2 function prevents eIF-2α phosphorylation, completely blocks reductions in translation, and inhibits gpdh-1 transcription. gpdh-1 expression is regulated by the highly conserved with-no-lysine kinase (WNK) and Ste20 kinases WNK-1 and GCK-3, which function in the GCN-2 signaling pathway downstream from eIF-2α phosphorylation. Our previous work has shown that hypertonic stress causes rapid and dramatic protein damage in C. elegans and that inhibition of translation reduces this damage. The current studies demonstrate that reduced translation also serves as an essential signal for activation of WNK-1/GCK-3 kinase signaling and subsequent transcription of gpdh-1 and possibly other osmoprotective genes.

  10. Genetic Variants in the STMN1 Transcriptional Regulatory Region Affect Promoter Activity and Fear Behavior in English Springer Spaniels.

    Directory of Open Access Journals (Sweden)

    Xiaolin Ding

    Full Text Available Stathmin 1 (STMN1 is a neuronal growth-associated protein that is involved in microtubule dynamics and plays an important role in synaptic outgrowth and plasticity. Given that STMN1 affects fear behavior, we hypothesized that genetic variations in the STMN1 transcriptional regulatory region affect gene transcription activity and control fear behavior. In this study, two single nucleotide polymorphisms (SNPs, g. -327 A>G and g. -125 C>T, were identified in 317 English Springer Spaniels. A bioinformatics analysis revealed that both were loci located in the canine STMN1 putative promoter region and affected transcription factor binding. A statistical analysis revealed that the TT genotype at g.-125 C>T produced a significantly greater fear level than that of the CC genotype (P < 0.05. Furthermore, the H4H4 (GTGT haplotype combination was significantly associated with canine fear behavior (P < 0.01. Using serially truncated constructs of the STMN1 promoters and the luciferase reporter, we found that a 395 bp (-312 nt to +83 nt fragment constituted the core promoter region. The luciferase assay also revealed that the H4 (GT haplotype promoter had higher activity than that of other haplotypes. Overall, our results suggest that the two SNPs in the canine STMN1 promoter region could affect canine fear behavior by altering STMN1 transcriptional activity.

  11. Peach MYB7 activates transcription of the proanthocyanidin pathway gene encoding leucoanthocyanidin reductase, but not anthocyanidin reductase

    Directory of Open Access Journals (Sweden)

    Hui eZhou

    2015-10-01

    Full Text Available Proanthocyanidins (PAs are a group of natural phenolic compounds that have a great effect on both flavour and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs via activation of PA-specific pathway genes encoding leucoanthocyanidin reductase (LAR and anthocyanidin reductase (ANR. Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5 via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants.

  12. Global analysis of transcription in castration-resistant prostate cancer cells uncovers active enhancers and direct androgen receptor targets.

    Science.gov (United States)

    Toropainen, Sari; Niskanen, Einari A; Malinen, Marjo; Sutinen, Päivi; Kaikkonen, Minna U; Palvimo, Jorma J

    2016-09-19

    Androgen receptor (AR) is a male sex steroid-activated transcription factor (TF) that plays a critical role in prostate cancers, including castration-resistant prostate cancers (CRPC) that typically express amplified levels of the AR. CRPC-derived VCaP cells display an excessive number of chromatin AR-binding sites (ARBs) most of which localize to distal inter- or intragenic regions. Here, we analyzed direct transcription programs of the AR in VCaP cells using global nuclear run-on sequencing (GRO-seq) and integrated the GRO-seq data with the ARB and VCaP cell-specific TF-binding data. Androgen immediately activated transcription of hundreds of protein-coding genes, including IGF-1 receptor and EGF receptor. Androgen also simultaneously repressed transcription of a large number of genes, including MYC. As functional enhancers have been postulated to produce enhancer-templated non-coding RNAs (eRNAs), we also analyzed the eRNAs, which revealed that only a fraction of the ARBs reside at functional enhancers. Activation of these enhancers was most pronounced at the sites that also bound PIAS1, ERG and HDAC3, whereas binding of HDAC3 and PIAS1 decreased at androgen-repressed enhancers. In summary, our genome-wide data of androgen-regulated enhancers and primary target genes provide new insights how the AR can directly regulate cellular growth and control signaling pathways in CPRC cells.

  13. High fat diet-induced changes of mouse hepatic transcription and enhancer activity can be reversed by subsequent weight loss.

    Science.gov (United States)

    Siersbæk, Majken; Varticovski, Lyuba; Yang, Shutong; Baek, Songjoon; Nielsen, Ronni; Mandrup, Susanne; Hager, Gordon L; Chung, Jay H; Grøntved, Lars

    2017-01-10

    Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome.

  14. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3.

    Science.gov (United States)

    Chew, Boon Shang; Siew, Wee Leng; Xiao, Benjamin; Lehming, Norbert

    2010-11-01

    Tbp1, the TATA-binding protein, is essential for transcriptional activation, and Gal4 and Gcn4 are unable to fully activate transcription in a Saccharomyces cerevisiae TBP1E86D mutant strain. In the present study we have shown that the Tbp1E186D mutant protein is proteolytically instable, and we have isolated intragenic and extragenic suppressors of the transcription defects of the TBP1E186D mutant strain. The TBP1R6S mutation stabilizes the Tbp1E186D mutant protein and suppresses the defects of the TBP1E186D mutant strain. Furthermore, we found that the overexpression of the de-ubiquitinating enzyme Ubp3 (ubiquitin-specific protease 3) also stabilizes the Tbp1E186D mutant protein and suppresses of the defects of the TBP1E186D mutant strain. Importantly, the deletion of UBP3 and its cofactor BRE5 lead to increased degradation of wild-type Tbp1 protein and to defects in transcriptional activation by Gal4 and Gcn4. Purified GST (glutathione transferase)-Ubp3 reversed Tbp1 ubiquitination, and the deletion of UBP3 lead to the accumulation of poly-ubiquitinated species of Tbp1 in a proteaseome-deficient genetic background, demonstrating that Ubp3 reverses ubiquitination of Tbp1 in vitro and in vivo. Chromatin immunoprecipitation showed that Ubp3 was recruited to the GAL1 and HIS3 promoters upon the induction of the respective gene, indicating that protection of promoter-bound Tbp1 by Ubp3 is required for transcriptional activation.

  15. Clonal selection for transcriptionally active viral oncogenes during progression to cancer.

    NARCIS (Netherlands)

    Tine, BA Van; Kappes, JC; Banerjee, NS; Knops, J; Lai, L; Steenbergen, R.D.M.; Meijer, C.J.L.M.; Snijders, P.J.F.; Chatis, P; Broker, TR; Moen, PTJr; Chow, L.T.

    2004-01-01

    Primary keratinocytes immortalized by human papillomaviruses (HPVs), along with HPV-induced cervical carcinoma cell lines, are excellent models for investigating neoplastic progression to cancer. By simultaneously visualizing viral DNA and nascent viral transcripts in interphase nuclei, we demonstra

  16. Nuclear Choline Acetyltransferase Activates Transcription of a High-affinity Choline Transporter*

    OpenAIRE

    Mats