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Sample records for e2f1 transcriptional activity

  1. E2F1 transcription is induced by genotoxic stress through ATM/ATR activation.

    PubMed

    Carcagno, Abel L; Ogara, María F; Sonzogni, Silvina V; Marazita, Mariela C; Sirkin, Pablo F; Ceruti, Julieta M; Cánepa, Eduardo T

    2009-05-01

    E2F1, a member of the E2F family of transcription factors, plays a critical role in controlling both cell cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. Following genotoxic stresses, E2F1 protein is stabilized by phosphorylation and acetylation driven to its accumulation. The aim of the present work was to examine whether the increase in E2F1 protein levels observed after DNA damage is only a reflection of an increase in E2F1 protein stability or is also the consequence of enhanced transcription of the E2F1 gene. The data presented here demonstrates that UV light and other genotoxics induce the transcription of E2F1 gene in an ATM/ATR dependent manner, which results in increasing E2F1 mRNA and protein levels. After genotoxic stress, transcription of cyclin E, an E2F1 target gene, was significantly induced. This induction was the result of two well-differentiated effects, one of them dependent on de novo protein synthesis and the other on the protein stabilization. Our results strongly support a transcriptional effect of DNA damaging agents on E2F1 expression. The results presented herein uncover a new mechanism involving E2F1 in response to genotoxic stress.

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

    SciTech Connect

    Kasim, Vivi, E-mail: vivikasim78@gmail.com; Huang, Can; Zhang, Jing

    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. Wemore » 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.« less

  3. E2F1 transcription factor and its impact on growth factor and cytokine signaling.

    PubMed

    Ertosun, Mustafa Gokhan; Hapil, Fatma Zehra; Osman Nidai, Ozes

    2016-10-01

    E2F1 is a transcription factor involved in cell cycle regulation and apoptosis. The transactivation capacity of E2F1 is regulated by pRb. In its hypophosphorylated form, pRb binds and inactivates DNA binding and transactivating functions of E2F1. The growth factor stimulation of cells leads to activation of CDKs (cyclin dependent kinases), which in turn phosphorylate Rb and hyperphosphorylated Rb is released from E2F1 or E2F1/DP complex, and free E2F1 can induce transcription of several genes involved in cell cycle entry, induction or inhibition of apoptosis. Thus, growth factors and cytokines generally utilize E2F1 to direct cells to either fate. Furthermore, E2F1 regulates expressions of various cytokines and growth factor receptors, establishing positive or negative feedback mechanisms. This review focuses on the relationship between E2F1 transcription factor and cytokines (IL-1, IL-2, IL-3, IL-6, TGF-beta, G-CSF, LIF), growth factors (EGF, KGF, VEGF, IGF, FGF, PDGF, HGF, NGF), and interferons (IFN-α, IFN-β and IFN-γ). Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. AMP-activated protein kinase α2 and E2F1 transcription factor mediate doxorubicin-induced cytotoxicity by forming a positive signal loop in mouse embryonic fibroblasts and non-carcinoma cells.

    PubMed

    Yang, Wookyeom; Park, In-Ja; Yun, Hee; Im, Dong-Uk; Ock, Sangmi; Kim, Jaetaek; Seo, Seon-Mi; Shin, Ha-Yeon; Viollet, Benoit; Kang, Insug; Choe, Wonchae; Kim, Sung-Soo; Ha, Joohun

    2014-02-21

    Doxorubicin is one of the most widely used anti-cancer drugs, but its clinical application is compromised by severe adverse effects in different organs including cardiotoxicity. In the present study we explored mechanisms of doxorubicin-induced cytotoxicity by revealing a novel role for the AMP-activated protein kinase α2 (AMPKα2) in mouse embryonic fibroblasts (MEFs). Doxorubicin robustly induced the expression of AMPKα2 in MEFs but slightly reduced AMPKα1 expression. Our data support the previous notion that AMPKα1 harbors survival properties under doxorubicin treatment. In contrast, analyses of Ampkα2(-/-) MEFs, gene knockdown of AMPKα2 by shRNA, and inhibition of AMPKα2 activity with an AMPK inhibitor indicated that AMPKα2 functions as a pro-apoptotic molecule under doxorubicin treatment. Doxorubicin induced AMPKα2 at the transcription level via E2F1, a transcription factor that regulates apoptosis in response to DNA damage. E2F1 directly transactivated the Ampkα2 gene promoter. In turn, AMPKα2 significantly contributed to stabilization and activation of E2F1 by doxorubicin, forming a positive signal amplification loop. AMPKα2 directly interacted with and phosphorylated E2F1. This signal loop was also detected in H9c2, C2C12, and ECV (human epithelial cells) cells as well as mouse liver under doxorubicin treatment. Resveratrol, which has been suggested to attenuate doxorubicin-induced cytotoxicity, significantly blocked induction of AMPKα2 and E2F1 by doxorubicin, leading to protection of these cells. This signal loop appears to be non-carcinoma-specific because AMPKα2 was not induced by doxorubicin in five different tested cancer cell lines. These results suggest that AMPKα2 may serve as a novel target for alleviating the cytotoxicity of doxorubicin.

  5. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    SciTech Connect

    Fang, Zejun; Gong, Chaoju; Liu, Hong

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression ofmore » E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration

  6. Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein.

    PubMed

    Sheldon, Lynn A

    2017-01-01

    The regulation of cell cycle progression by steroid hormones and growth factors is important for maintaining normal cellular processes including development and cell proliferation. Deregulated progression through the G1/S and G2/M cell cycle transitions can lead to uncontrolled cell proliferation and cancer. The transcription factor E2F1, a key cell cycle regulator, targets genes encoding proteins that regulate cell cycle progression through the G1/S transition as well as proteins important in DNA repair and apoptosis. E2F1 expression and activity is inhibited by inorganic arsenic (iAs) that has a dual role as a cancer therapeutic and as a toxin that leads to diseases including cancer. An understanding of what underlies this dichotomy will contribute to understanding how to use iAs as a more effective therapeutic and also how to treat cancers that iAs promotes. Here, we show that quiescent breast adenocarcinoma MCF-7 cells treated with 17-β estradiol (E2) progress through the cell cycle, but few cells treated with E2 + iAs progress from G1 into S-phase due to a block in cell cycle progression. Our data support a model in which iAs inhibits the dissociation of E2F1 from the tumor suppressor, retinoblastoma protein (pRB) due to changes in pRB phosphorylation which leads to decreased E2F1 transcriptional activity. These findings present an explanation for how iAs can disrupt cell cycle progression through E2F1-pRB and has implications for how iAs acts as a cancer therapeutic as well as how it may promote tumorigenesis through decreased DNA repair.

  7. HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

    PubMed

    Huang, Yuehua; Tai, Andrew W; Tong, Shuping; Lok, Anna S F

    2013-06-01

    Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  8. Bim, a Proapoptotic Protein, Up-regulated via Transcription Factor E2F1-dependent Mechanism, Functions as a Prosurvival Molecule in Cancer*

    PubMed Central

    Gogada, Raghu; Yadav, Neelu; Liu, Junwei; Tang, Shaohua; Zhang, Dianmu; Schneider, Andrea; Seshadri, Athul; Sun, Leimin; Aldaz, C. Marcelo; Tang, Dean G.; Chandra, Dhyan

    2013-01-01

    Proapoptotic Bcl-2 homology 3-only protein Bim plays an important role in Bax/Bak-mediated cytochrome c release and apoptosis. Here, we provide evidence for a novel prosurvival function of Bim in cancer cells. Bim was constitutively overexpressed in multiple prostate and breast cancer cells as well as in primary tumor cells. Quantitative real time PCR analysis showed that Bim was transcriptionally up-regulated. We have identified eight endogenous E2F1-binding sites on the Bim promoter using in silico analysis. Luciferase assay demonstrated that Bim expression was E2F1-dependent as mutation of the E2F1-binding sites on the Bim promoter inhibited luciferase activities. In support, E2F1 silencing led to the loss of Bim expression in cancer cells. Bim primarily localized to mitochondrial and cytoskeleton-associated fractions. Bim silencing or microinjection of anti-Bim antibodies into the cell cytoplasm resulted in cell rounding, detachment, and subsequent apoptosis. We observed up-regulation of prosurvival proteins Bcl-xL and Mcl-1, which sequester Bim in cancer cells. In addition, a phosphorylated form of Bim was also elevated in cancer cells. These findings suggest that the constitutively overexpressed Bim may function as a prosurvival molecule in epithelial cancer cells, and phosphorylation and association with Bcl-xL/Mcl-1 block its proapoptotic functions. PMID:23152504

  9. Functional synergy between DP-1 and E2F-1 in the cell cycle-regulating transcription factor DRTF1/E2F.

    PubMed Central

    Bandara, L R; Buck, V M; Zamanian, M; Johnston, L H; La Thangue, N B

    1993-01-01

    It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin-dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb-related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c-myc and DHFR. Two sequence-specific DNA binding proteins, E2F-1 and DP-1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP-1 and E2F-1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP-1 and E2F-1 interact synergistically in E2F site-dependent transcriptional activation. Images PMID:8223441

  10. Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor

    PubMed Central

    Kachhap, Sushant K.; Rosmus, Nadine; Collis, Spencer J.; Kortenhorst, Madeleine S. Q.; Wissing, Michel D.; Hedayati, Mohammad; Shabbeer, Shabana; Mendonca, Janet; Deangelis, Justin; Marchionni, Luigi; Lin, Jianqing; Höti, Naseruddin; Nortier, Johan W. R.; DeWeese, Theodore L.; Hammers, Hans; Carducci, Michael A.

    2010-01-01

    Background Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. Methodology/Principal Findings Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. Conclusions/Significance Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could

  11. The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

    PubMed Central

    Marlowe, Jennifer L.; Fan, Yunxia; Chang, Xiaoqing; Peng, Li; Knudsen, Erik S.; Xia, Ying

    2008-01-01

    Cellular stress by DNA damage induces checkpoint kinase-2 (CHK2)-mediated phosphorylation and stabilization of the E2F1 transcription factor, leading to induction of apoptosis by activation of a subset of proapoptotic E2F1 target genes, including Apaf1 and p73. This report characterizes an interaction between the aryl hydrocarbon (Ah) receptor (AHR), a ligand-activated transcription factor, and E2F1 that results in the attenuation of E2F1-mediated apoptosis. In Ahr−/− fibroblasts stably transfected with a doxycycline-regulated AHR expression vector, inhibition of AHR expression causes a significant elevation of oxidative stress, γH2A.X histone phosphorylation, and E2F1-dependent apoptosis, which can be blocked by small interfering RNA-mediated knockdown of E2F1 expression. In contrast, ligand-dependent AHR activation protects these cells from etoposide-induced cell death. In cells expressing both proteins, AHR and E2F1 interact independently of the retinoblastoma protein (RB), because AHR and E2F1 coimmunoprecipitate from extracts of RB-negative cells. Additionally, chromatin immunoprecipitation assays indicate that AHR and E2F1 bind to the Apaf1 promoter at a region containing a consensus E2F1 binding site but no AHR binding sites. AHR activation represses Apaf1 and TAp73 mRNA induction by a constitutively active CHK2 expression vector. Furthermore, AHR overexpression blocks the transcriptional induction of Apaf1 and p73 and the accumulation of sub-G0/G1 cells resulting from ectopic overexpression of E2F1. These results point to a proproliferative, antiapoptotic function of the Ah receptor that likely plays a role in tumor progression. PMID:18524851

  12. Bioinformatic detection of E47, E2F1 and SREBP1 transcription factors as potential regulators of genes associated to acquisition of endometrial receptivity

    PubMed Central

    2011-01-01

    Background The endometrium is a dynamic tissue whose changes are driven by the ovarian steroidal hormones. Its main function is to provide an adequate substrate for embryo implantation. Using microarray technology, several reports have provided the gene expression patterns of human endometrial tissue during the window of implantation. However it is required that biological connections be made across these genomic datasets to take full advantage of them. The objective of this work was to perform a research synthesis of available gene expression profiles related to acquisition of endometrial receptivity for embryo implantation, in order to gain insights into its molecular basis and regulation. Methods Gene expression datasets were intersected to determine a consensus endometrial receptivity transcript list (CERTL). For this cluster of genes we determined their functional annotations using available web-based databases. In addition, promoter sequences were analyzed to identify putative transcription factor binding sites using bioinformatics tools and determined over-represented features. Results We found 40 up- and 21 down-regulated transcripts in the CERTL. Those more consistently increased were C4BPA, SPP1, APOD, CD55, CFD, CLDN4, DKK1, ID4, IL15 and MAP3K5 whereas the more consistently decreased were OLFM1, CCNB1, CRABP2, EDN3, FGFR1, MSX1 and MSX2. Functional annotation of CERTL showed it was enriched with transcripts related to the immune response, complement activation and cell cycle regulation. Promoter sequence analysis of genes revealed that DNA binding sites for E47, E2F1 and SREBP1 transcription factors were the most consistently over-represented and in both up- and down-regulated genes during the window of implantation. Conclusions Our research synthesis allowed organizing and mining high throughput data to explore endometrial receptivity and focus future research efforts on specific genes and pathways. The discovery of possible new transcription factors

  13. Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis.

    PubMed

    Hou, Sheng T; Xie, Xiaoqi; Baggley, Anne; Park, David S; Chen, Gao; Walker, Teena

    2002-12-13

    Aberrant activation of the Rb/E2F1 pathway in cycling cells, in response to mitogenic or nonmitogenic stress signals, leads to apoptosis through hyperphosphorylation of Rb. To test whether in postmitotic neurons the Rb/E2F1 pathway can be activated by the nonmitogenic stress signaling, we examined the role of the p38 stress-activated protein kinase (SAPK) in regulating Rb phosphorylation in response to Fas (CD95/APO1)-mediated apoptosis of cultured cerebellar granule neurons (CGNs). Anti-Fas antibody induced a dramatic and early activation of p38. Activated p38 was correlated with the induction of hyperphosphorylation of both endogenous and exogenous Rb. The p38-selective inhibitor, SB203580, attenuated such an increase in pRb phosphorylation and significantly protected CGNs from Fas-induced apoptosis. The cyclin-dependent kinase-mediated Rb phosphorylation played a lesser role in this neuronal death paradigm, since cyclin-dependent kinase inhibitors, such as olomoucine, roscovitine, and flavopiridol, did not significantly prevent anti-Fas antibody-evoked neuronal apoptosis. Hyperphosphorylation of Rb by p38 SAPK resulted in the release of Rb-bound E2F1. Increased E2F1 modulated neuronal apoptosis, since E2F1-/- CGNs were significantly less susceptible to Fas-mediated apoptosis in comparison with the wild-type CGNs. Taken together, these studies demonstrate that neuronal Rb/E2F1 is modulated by the nonproliferative p38 SAPK in Fas-mediated neuronal apoptosis.

  14. CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes

    PubMed Central

    Singh, Randeep K.; Dagnino, Lina

    2017-01-01

    The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation. PMID:27903963

  15. CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes.

    PubMed

    Singh, Randeep K; Dagnino, Lina

    2017-01-17

    The E2F1 transcription factor plays key roles in skin homeostasis. In the epidermis, E2F1 expression is essential for normal proliferation of undifferentiated keratinocytes, regeneration after injury and DNA repair following UV radiation-induced photodamage. Abnormal E2F1 expression promotes nonmelanoma skin carcinoma. In addition, E2F1 must be downregulated for proper keratinocyte differentiation, but the relevant mechanisms involved remain poorly understood. We show that differentiation signals induce a series of post-translational modifications in E2F1 that are jointly required for its downregulation. Analysis of the structural determinants that govern these processes revealed a central role for S403 and T433. In particular, substitution of these two amino acid residues with non-phosphorylatable alanine (E2F1 ST/A) interferes with E2F1 nuclear export, K11- and K48-linked polyubiquitylation and degradation in differentiated keratinocytes. In contrast, replacement of S403 and T433 with phosphomimetic aspartic acid to generate a pseudophosphorylated E2F1 mutant protein (E2F1 ST/D) generates a protein that is regulated in a manner indistinguishable from that of wild type E2F1. Cdh1 is an activating cofactor that interacts with the anaphase-promoting complex/cyclosome (APC/C) ubiquitin E3 ligase, promoting proteasomal degradation of various substrates. We found that Cdh1 associates with E2F1 in keratinocytes. Inhibition or RNAi-mediated silencing of Cdh1 prevents E2F1 degradation in response to differentiation signals. Our results reveal novel regulatory mechanisms that jointly modulate post-translational modifications and downregulation of E2F1, which are necessary for proper epidermal keratinocyte differentiation.

  16. Nicotine, IFN-γ and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades.

    PubMed

    Kunigal, Sateesh; Ponnusamy, Moorthy P; Momi, Navneet; Batra, Surinder K; Chellappan, Srikumar P

    2012-04-26

    The membrane-bound mucins are thought to play an important biological role in cell-cell and cell-matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3. Chromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer. Our studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that targeting these signaling pathways

  17. Nicotine, IFN-γ and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades

    PubMed Central

    2012-01-01

    Background The membrane-bound mucins are thought to play an important biological role in cell–cell and cell–matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3. Results Chromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer. Conclusions Our studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that

  18. E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

    PubMed

    Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

    2016-11-01

    Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

  19. E2f1–3 Are Critical for Myeloid Development*

    PubMed Central

    Trikha, Prashant; Sharma, Nidhi; Opavsky, Rene; Reyes, Andres; Pena, Clarissa; Ostrowski, Michael C.; Roussel, Martine F.; Leone, Gustavo

    2011-01-01

    Hematopoietic development involves the coordinated activity of differentiation and cell cycle regulators. In current models of mammalian cell cycle control, E2f activators (E2f1, E2f2, and E2f3) are portrayed as the ultimate transcriptional effectors that commit cells to enter and progress through S phase. Using conditional gene knock-out strategies, we show that E2f1–3 are not required for the proliferation of early myeloid progenitors. Rather, these E2fs are critical for cell survival and proliferation at two distinct steps of myeloid development. First, E2f1–3 are required as transcriptional repressors for the survival of CD11b+ myeloid progenitors, and then they are required as activators for the proliferation of CD11b+ macrophages. In bone marrow macrophages, we show that E2f1–3 respond to CSF1-Myc mitogenic signals and serve to activate E2f target genes and promote their proliferation. Together, these findings expose dual functions for E2f1–3 at distinct stages of myeloid development in vivo, first as repressors in cell survival and then as activators in cell proliferation. In summary, this work places E2f1–3 in a specific signaling cascade that is critical for myeloid development in vivo. PMID:21115501

  20. The over expression of long non-coding RNA ANRIL promotes epithelial-mesenchymal transition by activating the ATM-E2F1 signaling pathway in pancreatic cancer: An in vivo and in vitro study.

    PubMed

    Chen, Shi; Zhang, Jia-Qiang; Chen, Jiang-Zhi; Chen, Hui-Xing; Qiu, Fu-Nan; Yan, Mao-Lin; Chen, Yan-Ling; Peng, Cheng-Hong; Tian, Yi-Feng; Wang, Yao-Dong

    2017-09-01

    This study aims to investigate the roles of lncRNA ANRIL in epithelial-mesenchymal transition (EMT) by regulating the ATM-E2F1 signaling pathway in pancreatic cancer (PC). PC rat models were established and ANRIL overexpression and interference plasmids were transfected. The expression of ANRIL, EMT markers (E-cadherin, N-cadherin and Vimentin) and ATM-E2F1 signaling pathway-related proteins (ATM, E2F1, INK4A, INK4B and ARF) were detected. Small molecule drugs were applied to activate and inhibit the ATM-E2F1 signaling pathway. Transwell assay and the scratch test were adopted to detect cell invasion and migration abilities. ANRIL expression in the PC cells was higher than in normal pancreatic duct epithelial cells. In the PC rat models and PC cells, ANRIL interference promoted the expressions of INK4B, INK4A, ARF and E-cadherin, while reduced N-cadherin and Vimentin expression. Over-expressed ANRIL decreased the expression of INK4B, INK4A, ARF and E-cadherin, but raised N-cadherin and Vimentin expressions. By inhibiting the ATM-E2F1 signaling pathway in PC cells, E-cadherin expression increased but N-cadherin and Vimentin expressions decreased. After ANRIL was silenced or the ATM-E2F1 signaling pathway inhibited, PC cell migration and invasion abilities were decreased. In conclusion, over-expression of lncRNA ANRIL can promote EMT of PC cells by activating the ATM-E2F1 signaling pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

    PubMed

    Zhou, Fangfang; Zhang, Long; Wang, Aijun; Song, Bo; Gong, Kai; Zhang, Lihai; Hu, Min; Zhang, Xiufang; Zhao, Nanming; Gong, Yandao

    2008-05-23

    It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

  2. A novel mechanism of E2F1 regulation via nucleocytoplasmic shuttling: determinants of nuclear import and export.

    PubMed

    Ivanova, Iordanka A; Vespa, Alisa; Dagnino, Lina

    2007-09-01

    E2F1 is a transcription factor central for cell survival, proliferation, and repair following genomic insult. Depending on the cell type and conditions, E2F1 can induce apoptosis in transformed cells, behaving as a tumour suppressor, or impart growth advantages favouring tumour formation. The pleiotropic functions of E2F1 are a likely consequence of its ability to transcriptionally control a wide variety of target genes, and require tight regulation of its activity at multiple levels. Although sequestration of proteins to particular cellular compartments is a well-established regulatory mechanism, virtually nothing is known about its contribution to modulation of E2F1 target gene expression. We have examined the subcellular trafficking of E2F1 and, contrary to the widely held notion that this factor is constitutively nuclear, we now demonstrate that it is subjected to continuous nucleocytoplasmic shuttling. We have also defined two nuclear localization domains and a nuclear export region, which mediates CRM1-dependent transit out of the nucleus. The predominant subcellular location of E2F1 is likely determined by the balance between the activity of nuclear import and export domains, and can be modulated by differentiation stimuli in epidermal cells. Thus, we have identified a hitherto unrecognized mechanism to control E2F1 function through modulation of its subcellular localization.

  3. MiR-205-5p and miR-342-3p cooperate in the repression of the E2F1 transcription factor in the context of anticancer chemotherapy resistance

    PubMed Central

    Lai, Xin; Gupta, Shailendra K; Schmitz, Ulf; Marquardt, Stephan; Knoll, Susanne; Spitschak, Alf; Wolkenhauer, Olaf; Pützer, Brigitte M; Vera, Julio

    2018-01-01

    High rates of lethal outcome in tumour metastasis are associated with the acquisition of invasiveness and chemoresistance. Several clinical studies indicate that E2F1 overexpression across high-grade tumours culminates in unfavourable prognosis and chemoresistance in patients. Thus, fine-tuning the expression of E2F1 could be a promising approach for treating patients showing chemoresistance. Methods: We integrated bioinformatics, structural and kinetic modelling, and experiments to study cooperative regulation of E2F1 by microRNA (miRNA) pairs in the context of anticancer chemotherapy resistance. Results: We showed that an enhanced E2F1 repression efficiency can be achieved in chemoresistant tumour cells through two cooperating miRNAs. Sequence and structural information were used to identify potential miRNA pairs that can form tertiary structures with E2F1 mRNA. We then employed molecular dynamics simulations to show that among the identified triplexes, miR-205-5p and miR-342-3p can form the most stable triplex with E2F1 mRNA. A mathematical model simulating the E2F1 regulation by the cooperative miRNAs predicted enhanced E2F1 repression, a feature that was verified by in vitro experiments. Finally, we integrated this cooperative miRNA regulation into a more comprehensive network to account for E2F1-related chemoresistance in tumour cells. The network model simulations and experimental data indicate the ability of enhanced expression of both miR-205-5p and miR-342-3p to decrease tumour chemoresistance by cooperatively repressing E2F1. Conclusions: Our results suggest that pairs of cooperating miRNAs could be used as potential RNA therapeutics to reduce E2F1-related chemoresistance. PMID:29464002

  4. ROS Production Is Essential for the Apoptotic Function of E2F1 in Pheochromocytoma and Neuroblastoma Cell Lines

    PubMed Central

    Espada, Lilia; Meo-Evoli, Nathalie; Sancho, Patricia; Real, Sebastian; Fabregat, Isabel; Ambrosio, Santiago; Tauler, Albert

    2012-01-01

    In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator

  5. ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage

    PubMed Central

    Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

    2014-01-01

    The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein–protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage. PMID:24675884

  6. ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

    PubMed

    Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

    2014-01-01

    The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

  7. Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1

    PubMed Central

    Zheng, Shunsheng; Moehlenbrink, Jutta; Lu, Yi-Chien; Zalmas, Lykourgos-Panagiotis; Sagum, Cari A.; Carr, Simon; McGouran, Joanna F.; Alexander, Leila; Fedorov, Oleg; Munro, Shonagh; Kessler, Benedikt; Bedford, Mark T.; Yu, Qiang; La Thangue, Nicholas B.

    2014-01-01

    Summary The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase (PRMT) 1 and symmetric dimethylating PRMT5, and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favours proliferation by antagonising methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN down-regulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity. PMID:24076217

  8. Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice.

    PubMed

    Iglesias, Ainhoa; Murga, Matilde; Laresgoiti, Usua; Skoudy, Anouchka; Bernales, Irantzu; Fullaondo, Asier; Moreno, Bernardino; Lloreta, José; Field, Seth J; Real, Francisco X; Zubiaga, Ana M

    2004-05-01

    E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

  9. Elevated autophagy gene expression in adipose tissue of obese humans: A potential non-cell-cycle-dependent function of E2F1

    PubMed Central

    Haim, Yulia; Blüher, Matthias; Slutsky, Noa; Goldstein, Nir; Klöting, Nora; Harman-Boehm, Ilana; Kirshtein, Boris; Ginsberg, Doron; Gericke, Martin; Guiu Jurado, Esther; Kovsan, Julia; Tarnovscki, Tanya; Kachko, Leonid; Bashan, Nava; Gepner, Yiftach; Shai, Iris; Rudich, Assaf

    2015-01-01

    Autophagy genes' expression is upregulated in visceral fat in human obesity, associating with obesity-related cardio-metabolic risk. E2F1 (E2F transcription factor 1) was shown in cancer cells to transcriptionally regulate autophagy. We hypothesize that E2F1 regulates adipocyte autophagy in obesity, associating with endocrine/metabolic dysfunction, thereby, representing non-cell-cycle function of this transcription factor. E2F1 protein (N=69) and mRNA (N=437) were elevated in visceral fat of obese humans, correlating with increased expression of ATG5 (autophagy-related 5), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β), but not with proliferation/cell-cycle markers. Elevated E2F1 mainly characterized the adipocyte fraction, whereas MKI67 (marker of proliferation Ki-67) was elevated in the stromal-vascular fraction of adipose tissue. In human visceral fat explants, chromatin-immunoprecipitation revealed body mass index (BMI)-correlated increase in E2F1 binding to the promoter of MAP1LC3B, but not to the classical cell cycle E2F1 target, CCND1 (cyclin D1). Clinically, omental fat E2F1 expression correlated with insulin resistance, circulating free-fatty-acids (FFA), and with decreased circulating ADIPOQ/adiponectin, associations attenuated by adjustment for autophagy genes. Overexpression of E2F1 in HEK293 cells enhanced promoter activity of several autophagy genes and autophagic flux, and sensitized to further activation of autophagy by TNF. Conversely, mouse embryonic fibroblast (MEF)-derived adipocytes from e2f1 knockout mice (e2f1−/−) exhibited lower autophagy gene expression and flux, were more insulin sensitive, and secreted more ADIPOQ. Furthermore, e2f1−/− MEF-derived adipocytes, and autophagy-deficient (by Atg7 siRNA) adipocytes were resistant to cytokines-induced decrease in ADIPOQ secretion. Jointly, upregulated E2F1 sensitizes adipose tissue autophagy to inflammatory stimuli, linking visceral obesity to adipose and systemic

  10. Copy number variations of E2F1: a new genetic risk factor for testicular cancer.

    PubMed

    Rocca, Maria Santa; Di Nisio, Andrea; Marchiori, Arianna; Ghezzi, Marco; Opocher, Giuseppe; Foresta, Carlo; Ferlin, Alberto

    2017-03-01

    Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1 Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer. © 2017 Society for Endocrinology.

  11. Interrupted E2F1-miR-34c-SCF negative feedback loop by hyper-methylation promotes colorectal cancer cell proliferation

    PubMed Central

    Yang, Shu; Wu, Bo; Sun, Haimei; Ji, Fengqing; Sun, Tingyi; Zhao, Yan; Zhou, Deshan

    2015-01-01

    Tumour suppressor miR-34c deficiency resulted from hyper-methylation in its promoter is believed to be one of the main causes of colorectal cancer (CRC). Till date, miR-34c has been validated as a direct target of p53; but previous evidence suggested other transcription factor(s) must be involved in miR-34c transcription. In the present study, we in the first place identified a core promoter region (−1118 to −883 bp) of pre-miR-34c which was embedded within a hyper-methylated CpG island. Secondly, E2F1 promoted miR-34c transcription by physical interaction with the miR-34c promoter at site −897 to −889 bp. The transcriptional activating effect of E2F1 on miR-34c was in a p53 independent manner but profoundly promoted in the presence of p53 with exposure to 5-aza-2′-deoxycytidine (DAC). Thirdly, stem cell factor (SCF), a miR-34c target, was specifically reduced upon an introduction of E2F1 which lead to suppression of CRC cell proliferation. The E2F1-suppressed cell proliferation was partially abrogated by additional miR-34c inhibitor, indicating that the anti-proliferation effect of E2F1 was probably through activating miR-34c-SCF axis. Finally, SCF/KIT signalling increased E2F1 production by reducing its proteosomal degradation dependent on PI3K/Akt-GSK3β pathway. In conclusion, our results suggested the existence of E2F1-miR-34c-SCF negative feedback loop which was interrupted by the hyper-methylation of miR-34c promoter in CRC cells and increased cell proliferation. PMID:26704889

  12. E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution.

    PubMed

    Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

    2015-10-01

    Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53(-/-) mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

  13. E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

    PubMed Central

    Iglesias-Ara, A; Zenarruzabeitia, O; Buelta, L; Merino, J; Zubiaga, A M

    2015-01-01

    Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53−/− mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy. PMID:25656653

  14. The prohibitin-repressive interaction with E2F1 is rapidly inhibited by androgen signalling in prostate cancer cells

    PubMed Central

    Koushyar, S; Economides, G; Zaat, S; Jiang, W; Bevan, C L; Dart, D A

    2017-01-01

    Prohibitin (PHB) is a tumour suppressor molecule with pleiotropic activities across several cellular compartments including mitochondria, cell membrane and the nucleus. PHB and the steroid-activated androgen receptor (AR) have an interplay where AR downregulates PHB, and PHB represses AR. Additionally, their cellular locations and chromatin interactions are in dynamic opposition. We investigated the mechanisms of cell cycle inhibition by PHB and how this is modulated by AR in prostate cancer. Using a prostate cancer cell line overexpressing PHB, we analysed the gene expression changes associated with PHB-mediated cell cycle arrest. Over 1000 gene expression changes were found to be significant and gene ontology analysis confirmed PHB-mediated repression of genes essential for DNA replication and synthesis, for example, MCMs and TK1, via an E2F1 regulated pathway—agreeing with its G1/S cell cycle arrest activity. PHB is known to inhibit E2F1-mediated transcription, and the PHB:E2F1 interaction was seen in LNCaP nuclear extracts, which was then reduced by androgen treatment. Upon two-dimensional western blot analysis, the PHB protein itself showed androgen-mediated charge differentiation (only in AR-positive cells), indicating a potential dephosphorylation event. Kinexus phosphoprotein array analysis indicated that Src kinase was the main interacting intracellular signalling hub in androgen-treated LNCaP cells, and that Src inhibition could reduce this AR-mediated charge differentiation. PHB charge change may be associated with rapid dissociation from chromatin and E2F1, allowing the cell cycle to proceed. The AR and androgens may deactivate the repressive functions of PHB upon E2F1 leading to cell cycle progression, and indicates a role for AR in DNA replication licensing. PMID:28504694

  15. KDM4A Coactivates E2F1 to Regulate the PDK-Dependent Metabolic Switch between Mitochondrial Oxidation and Glycolysis.

    PubMed

    Wang, Ling-Yu; Hung, Chiu-Lien; Chen, Yun-Ru; Yang, Joy C; Wang, Junjian; Campbell, Mel; Izumiya, Yoshihiro; Chen, Hong-Wu; Wang, Wen-Ching; Ann, David K; Kung, Hsing-Jien

    2016-09-13

    The histone lysine demethylase KDM4A/JMJD2A has been implicated in prostate carcinogenesis through its role in transcriptional regulation. Here, we describe KDM4A as a E2F1 coactivator and demonstrate a functional role for the E2F1-KDM4A complex in the control of tumor metabolism. KDM4A associates with E2F1 on target gene promoters and enhances E2F1 chromatin binding and transcriptional activity, thereby modulating the transcriptional profile essential for cancer cell proliferation and survival. The pyruvate dehydrogenase kinases (PDKs) PDK1 and PDK3 are direct targets of KDM4A and E2F1 and modulate the switch between glycolytic metabolism and mitochondrial oxidation. Downregulation of KDM4A leads to elevated activity of pyruvate dehydrogenase and mitochondrial oxidation, resulting in excessive accumulation of reactive oxygen species. The altered metabolic phenotypes can be partially rescued by ectopic expression of PDK1 and PDK3, indicating a KDM4A-dependent tumor metabolic regulation via PDK. Our results suggest that KDM4A is a key regulator of tumor metabolism and a potential therapeutic target for prostate cancer. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. E2F1 and NF-κB: Key Mediators of Inflammation-associated Cancers and Potential Therapeutic Targets.

    PubMed

    Huang, Yulin; Chen, Rui; Zhou, Jianwei

    2016-01-01

    Inflammation is the fundamental protective response; however disordered immuno-response can cause chronic human disease, including cancer. Inflammatory cells and mediators are essential to the tumor microenvironment and dissection of this complex molecular and cellular milieu may elucidate a connection between cancer and inflammation and help to identify potential novel therapeutic targets. Thus, focusing on transcription factor NF-κB and E2F1 in inflammation-associated cancer is urgent. NF-κB activation is prevalent in carcinomas, mainly driven by inflammatory cytokines in the tumor microenvironment. E2F1 is also involved in regulating immune responses. Understanding the crosstalk between the two pathways may contribute to the development of novel anti-cancer drugs.

  17. E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair.

    PubMed

    Singh, Randeep K; Dagnino, Lina

    2016-05-03

    Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis.

  18. E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair

    PubMed Central

    Singh, Randeep K.; Dagnino, Lina

    2016-01-01

    Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis. PMID:27028861

  19. HER2 signaling drives DNA anabolism and proliferation through SRC-3 phosphorylation and E2F1-regulated genes

    PubMed Central

    Nikolai, Bryan C.; Lanz, Rainer B.; York, Brian; Dasgupta, Subhamoy; Mitsiades, Nicholas; Creighton, Chad J.; Tsimelzon, Anna; Hilsenbeck, Susan G.; Lonard, David M.; Smith, Carolyn L.; O’Malley, Bert W.

    2016-01-01

    Approximately 20% of early-stage breast cancers display amplification or overexpression of the ErbB2/HER2 oncogene, conferring poor prognosis and resistance to endocrine therapy. Targeting HER2+ tumors with trastuzumab or the receptor tyrosine kinase (RTK) inhibitor lapatinib significantly improves survival, yet tumor resistance and progression of metastatic disease still develop over time. While the mechanisms of cytosolic HER2 signaling are well studied, nuclear signaling components and gene regulatory networks that bestow therapeutic resistance and limitless proliferative potential are incompletely understood. Here, we use biochemical and bioinformatic approaches to identify effectors and targets of HER2 transcriptional signaling in human breast cancer. Phosphorylation and activity of the Steroid Receptor Coactivator-3 (SRC-3) is reduced upon HER2 inhibition, and recruitment of SRC-3 to regulatory elements of endogenous genes is impaired. Transcripts regulated by HER2 signaling are highly enriched with E2F1 binding sites and define a gene signature associated with proliferative breast tumor subtypes, cell cycle progression, and DNA replication. We show that HER2 signaling promotes breast cancer cell proliferation through regulation of E2F1-driven DNA metabolism and replication genes together with phosphorylation and activity of the transcriptional coactivator SRC-3. Furthermore, our analyses identified a cyclin dependent kinase (CDK) signaling node that, when targeted using the CDK4/6 inhibitor Palbociclib, defines overlap and divergence of adjuvant pharmacological targeting. Importantly, lapatinib and palbociclib strictly block de novo synthesis of DNA, mostly through disruption of E2F1 and its target genes. These results have implications for rational discovery of pharmacological combinations in pre-clinical models of adjuvant treatment and therapeutic resistance. PMID:26833126

  20. ERBB2 Deficiency Alters an E2F-1-Dependent Adaptive Stress Response and Leads to Cardiac Dysfunction

    PubMed Central

    Perry, Marie-Claude; Dufour, Catherine R.; Eichner, Lillian J.; Tsang, David W. K.; Deblois, Geneviève; Muller, William J.

    2014-01-01

    The tyrosine kinase receptor ERBB2 is required for normal development of the heart and is a potent oncogene in breast epithelium. Trastuzumab, a monoclonal antibody targeting ERBB2, improves the survival of breast cancer patients, but cardiac dysfunction is a major side effect of the drug. The molecular mechanisms underlying how ERBB2 regulates cardiac function and why trastuzumab is cardiotoxic remain poorly understood. We show here that ERBB2 hypomorphic mice develop cardiac dysfunction that mimics the side effects observed in patients treated with trastuzumab. We demonstrate that this phenotype is related to the critical role played by ERBB2 in cardiac homeostasis and physiological hypertrophy. Importantly, genetic and therapeutic reduction of ERBB2 activity in mice, as well as ablation of ERBB2 signaling by trastuzumab or siRNAs in human cardiomyocytes, led to the identification of an impaired E2F-1-dependent genetic program critical for the cardiac adaptive stress response. These findings demonstrate the existence of a previously unknown mechanistic link between ERBB2 and E2F-1 transcriptional activity in heart physiology and trastuzumab-induced cardiac dysfunction. PMID:25246633

  1. E2F1/TS Immunophenotype and Survival of Patients with Colorectal Cancer Treated with 5FU-Based Adjuvant Therapy.

    PubMed

    Sulzyc-Bielicka, Violetta; Domagala, Pawel; Bielicki, Dariusz; Safranow, Krzysztof; Rogowski, Wojciech; Domagala, Wenancjusz

    2016-07-01

    The predictive value of thymidylate synthase (TS) expression alone for 5FU-based treatment of colorectal cancer (CRC) has not been clinically confirmed. Little is known on the association of expression of E2F1, which controls the transcription of genes encoding proteins engaged in DNA synthesis including TS, and survival of patients with CRC. The purpose of this study is to assess the correlation between expression of both E2F1 and TS in CRCs and survival of patients administered adjuvant 5FU-based chemotherapy, in order to find a better predictor of treatment outcome than expression of TS or E2F1 alone. Nuclear TS and E2F1 were detected by immunohistochemistry in tissue microarrays from 190 CRCs (Astler-Coller stage B2 or C). Multivariate analysis identified significant association of the combined E2F1+TS+ immunophenotype with worse OS (HR = 3,78, P = 0,009) and DFS (HR = 2,30, P = 0,03) of patients with colon cancer. There were significant differences between E2F1+TS+ and E2F1-TS- Kaplan-Meier survival curves in relation to DFS (P = 0.008) and OS (P = 0.01). About 37 and 31 % difference in 3-year DFS and OS respectively were seen between patients with E2F1+TS+ vs. E2F1-TS- colon cancer immunophenotype. The E2F1+TS+ immunophenotype may be a marker of poor prognosis (the worst DFS and OS) of patients with colon cancer treated with 5FU-based adjuvant therapy. A subgroup of patients with this immunophenotype may require different and perhaps more aggressive treatment than 5FU-based chemotherapy. Thus, the combined E2F1/TS immunophenotype could be a potential indicator of colon cancer sensitivity to 5FU.

  2. E2F1 somatic mutation within miRNA target site impairs gene regulation in colorectal cancer.

    PubMed

    Lopes-Ramos, Camila M; Barros, Bruna P; Koyama, Fernanda C; Carpinetti, Paola A; Pezuk, Julia; Doimo, Nayara T S; Habr-Gama, Angelita; Perez, Rodrigo O; Parmigiani, Raphael B

    2017-01-01

    Genetic studies have largely concentrated on the impact of somatic mutations found in coding regions, and have neglected mutations outside of these. However, 3' untranslated regions (3' UTR) mutations can also disrupt or create miRNA target sites, and trigger oncogene activation or tumor suppressor inactivation. We used next-generation sequencing to widely screen for genetic alterations within predicted miRNA target sites of oncogenes associated with colorectal cancer, and evaluated the functional impact of a new somatic mutation. Target sequencing of 47 genes was performed for 29 primary colorectal tumor samples. For 71 independent samples, Sanger methodology was used to screen for E2F1 mutations in miRNA predicted target sites, and the functional impact of these mutations was evaluated by luciferase reporter assays. We identified germline and somatic alterations in E2F1. Of the 100 samples evaluated, 3 had germline alterations at the MIR205-5p target site, while one had a somatic mutation at MIR136-5p target site. E2F1 gene expression was similar between normal and tumor tissues bearing the germline alteration; however, expression was increased 4-fold in tumor tissue that harbored a somatic mutation compared to that in normal tissue. Luciferase reporter assays revealed both germline and somatic alterations increased E2F1 activity relative to wild-type E2F1. We demonstrated that somatic mutation within E2F1:MIR136-5p target site impairs miRNA-mediated regulation and leads to increased gene activity. We conclude that somatic mutations that disrupt miRNA target sites have the potential to impact gene regulation, highlighting an important mechanism of oncogene activation.

  3. E2F1-mediated upregulation of p19INK4d determines its periodic expression during cell cycle and regulates cellular proliferation.

    PubMed

    Carcagno, Abel L; Marazita, Mariela C; Ogara, María F; Ceruti, Julieta M; Sonzogni, Silvina V; Scassa, María E; Giono, Luciana E; Cánepa, Eduardo T

    2011-01-01

    A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an additional mechanism to limit E2F activity.

  4. E2F1-Mediated Upregulation of p19INK4d Determines Its Periodic Expression during Cell Cycle and Regulates Cellular Proliferation

    PubMed Central

    Carcagno, Abel L.; Marazita, Mariela C.; Ogara, María F.; Ceruti, Julieta M.; Sonzogni, Silvina V.; Scassa, María E.; Giono, Luciana E.; Cánepa, Eduardo T.

    2011-01-01

    Background A central aspect of development and disease is the control of cell proliferation through regulation of the mitotic cycle. Cell cycle progression and directionality requires an appropriate balance of positive and negative regulators whose expression must fluctuate in a coordinated manner. p19INK4d, a member of the INK4 family of CDK inhibitors, has a unique feature that distinguishes it from the remaining INK4 and makes it a likely candidate for contributing to the directionality of the cell cycle. p19INK4d mRNA and protein levels accumulate periodically during the cell cycle under normal conditions, a feature reminiscent of cyclins. Methodology/Principal Findings In this paper, we demonstrate that p19INK4d is transcriptionally regulated by E2F1 through two response elements present in the p19INK4d promoter. Ablation of this regulation reduced p19 levels and restricted its expression during the cell cycle, reflecting the contribution of a transcriptional effect of E2F1 on p19 periodicity. The induction of p19INK4d is delayed during the cell cycle compared to that of cyclin E, temporally separating the induction of these proliferative and antiproliferative target genes. Specific inhibition of the E2F1-p19INK4d pathway using triplex-forming oligonucleotides that block E2F1 binding on p19 promoter, stimulated cell proliferation and increased the fraction of cells in S phase. Conclusions/Significance The results described here support a model of normal cell cycle progression in which, following phosphorylation of pRb, free E2F induces cyclin E, among other target genes. Once cyclinE/CDK2 takes over as the cell cycle driving kinase activity, the induction of p19 mediated by E2F1 leads to inhibition of the CDK4,6-containing complexes, bringing the G1 phase to an end. This regulatory mechanism constitutes a new negative feedback loop that terminates the G1 phase proliferative signal, contributing to the proper coordination of the cell cycle and provides an

  5. TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer.

    PubMed

    Ma, Yueyun; Xin, Yijuan; Li, Rui; Wang, Zhe; Yue, Qiaohong; Xiao, Fengjing; Hao, Xiaoke

    2014-03-10

    TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors

    PubMed Central

    Kim, So Yoon; Rane, Sushil G.

    2011-01-01

    Cell division and cell differentiation are intricately regulated processes vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrate the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions, whereas Cdks 2, 4 and 6 are dispensable for organogenesis but vital for tissue-specific cell development. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the epithelium to give rise to the distinct cell lineages of the adult pancreas. The cell cycle molecules that specify lineage commitment within the early pancreas are unknown. We show that Cdk4 and its downstream transcription factor E2f1 regulate mouse pancreas development prior to and during the secondary transition. Cdk4 deficiency reduces embryonic pancreas size owing to impaired mesenchyme development and fewer Pdx1+ pancreatic progenitor cells. Expression of activated Cdk4R24C kinase leads to increased Nkx2.2+ and Nkx6.1+ cells and a rise in the number and proliferation of Ngn3+ endocrine precursors, resulting in expansion of the β cell lineage. We show that E2f1 binds and activates the Ngn3 promoter to modulate Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes β cell development by directing E2f1-mediated activation of Ngn3 and increasing the pool of endocrine precursors, and identify Cdk4 as an important regulator of early pancreas development that modulates the proliferation potential of pancreatic progenitors and endocrine precursors. PMID:21490060

  7. Augmentation of the therapeutic efficacy of WEE1 kinase inhibitor AZD1775 by inhibiting the YAP-E2F1-DNA damage response pathway axis.

    PubMed

    Oku, Yusuke; Nishiya, Naoyuki; Tazawa, Takaaki; Kobayashi, Takaya; Umezawa, Nanami; Sugawara, Yasuyo; Uehara, Yoshimasa

    2018-06-01

    The main reasons for failure of cancer chemotherapy are intrinsic and acquired drug resistance. The Hippo pathway effector Yes-associated protein (YAP) is associated with resistance to both cytotoxic and molecular targeted drugs. Several lines of evidence indicate that YAP activates transcriptional programmes to promote cell cycle progression and DNA damage responses. Therefore, we hypothesised that YAP is involved in the sensitivity of cancer cells to small-molecule agents targeting cell cycle-related proteins. Here, we report that the inactivation of YAP sensitises the OVCAR-8 ovarian cancer cell line to AZD1775, a small-molecule WEE1 kinase inhibitor. The accumulation of DNA damage and mitotic failures induced by AZD1775-based therapy were further enhanced by YAP depletion. YAP depletion reduced the expression of the Fanconi anaemia (FA) pathway components required for DNA repair and their transcriptional regulator E2F1. These results suggest that YAP activates the DNA damage response pathway, exemplified by the FA pathway and E2F1. Furthermore, we aimed to apply this finding to combination chemotherapy against ovarian cancers. The regimen containing dasatinib, which inhibits the nuclear localisation of YAP, improved the response to AZD1775-based therapy in the OVCAR-8 ovarian cancer cell line. We propose that dasatinib acts as a chemosensitiser for a subset of molecular targeted drugs, including AZD1775, by targeting YAP.

  8. E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

    PubMed

    Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

    2015-01-01

    E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

  9. E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells

    PubMed Central

    Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

    2015-01-01

    E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells. PMID:25892555

  10. THE ROLE OF THE RETINOBLASTOMA/E2F1 TUMOR SUPPRESSOR PATHWAY IN THE LESION RECOGNITION STEP OF NUCLEOTIDE EXCISION REPAIR

    PubMed Central

    Lin, Patrick S.; McPherson, Lisa A.; Chen, Aubrey Y.; Sage, Julien; Ford, James M.

    2009-01-01

    The retinoblastoma Rb/E2F tumor suppressor pathway plays a major role in the regulation of mammalian cell cycle progression. The pRb protein, along with closely related proteins p107 and p130, exerts its anti-proliferative effects by binding to the E2F family of transcription factors known to regulate essential genes throughout the cell cycle. We sought to investigate the role of the Rb/E2F1 pathway in the lesion recognition step of nucleotide excision repair (NER) in mouse embryonic fibroblasts (MEFs). Rb−/−;p107−/−;p130−/− MEFs repaired both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PPs) at higher efficiency than did wildtype cells following UV-C irradiation. The expression of damaged DNA binding gene DDB2 involved in the DNA lesion recognition step was elevated in the Rb family-deficient MEFs. To determine if the enhanced DNA repair in the absence of the Rb gene family is due to the derepression of E2F1, we assayed the ability of E2F1-deficient cells to repair damaged DNA and demonstrated that E2F1−/− MEFs are impaired for the removal of both CPDs and 6-4PPs. Furthermore, wildtype cells induced a higher expression of DDB2 and xeroderma pigmentosum gene XPC transcript levels than did E2F1−/− cells following UV-C irradiation. Using an E2F SiteScan algorithm, we uncovered a putative E2F-responsive element in the XPC promoter upstream of the transcription start site. We showed with chromatin immunoprecipitation assays the binding of E2F1 to the XPC promoter in a UV-dependent manner, suggesting that E2F1 is a transcriptional regulator of XPC. Our study identifies a novel E2F1 gene target and further supports the growing body of evidence that the Rb/E2F1 tumor suppressor pathway is involved in the regulation of the DNA lesion recognition step of nucleotide excision repair. PMID:19376752

  11. First somatic mutation of E2F1 in a critical DNA binding residue discovered in well-differentiated papillary mesothelioma of the peritoneum

    PubMed Central

    2011-01-01

    Background Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP. Results WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector. Conclusions The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1. PMID:21955916

  12. E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

    PubMed

    Carcagno, Abel L; Giono, Luciana E; Marazita, Mariela C; Castillo, Daniela S; Pregi, Nicolás; Cánepa, Eduardo T

    2012-07-01

    Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.

  13. Loss of GCN5 leads to increased neuronal apoptosis by upregulating E2F1- and Egr-1-dependent BH3-only protein Bim.

    PubMed

    Wu, Yanna; Ma, Shanshan; Xia, Yong; Lu, Yangpeng; Xiao, Shiyin; Cao, Yali; Zhuang, Sidian; Tan, Xiangpeng; Fu, Qiang; Xie, Longchang; Li, Zhiming; Yuan, Zhongmin

    2017-01-26

    Cellular acetylation homeostasis is a kinetic balance precisely controlled by histone acetyl-transferase (HAT) and histone deacetylase (HDAC) activities. The loss of the counterbalancing function of basal HAT activity alters the precious HAT:HDAC balance towards enhanced histone deacetylation, resulting in a loss of acetylation homeostasis, which is closely associated with neuronal apoptosis. However, the critical HAT member whose activity loss contributes to neuronal apoptosis remains to be identified. In this study, we found that inactivation of GCN5 by either pharmacological inhibitors, such as CPTH2 and MB-3, or by inactivation with siRNAs leads to a typical apoptosis in cultured cerebellar granule neurons. Mechanistically, the BH3-only protein Bim is transcriptionally upregulated by activated Egr-1 and E2F1 and mediates apoptosis following GCN5 inhibition. Furthermore, in the activity withdrawal- or glutamate-evoked neuronal apoptosis models, GCN5 loses its activity, in contrast to Bim induction. Adenovirus-mediated overexpression of GCN5 suppresses Bim induction and apoptosis. Interestingly, the loss of GCN5 activity and the induction of Egr-1, E2F1 and Bim are involved in the early brain injury (EBI) following subarachnoid haemorrhage (SAH) in rats. HDAC inhibition not only significantly rescues Bim expression and apoptosis induced by either potassium deprivation or GCN5 inactivation but also ameliorates these events and EBI in SAH rats. Taken together, our results highlight a new mechanism by which the loss of GCN5 activity promotes neuronal apoptosis through the transcriptional upregulation of Bim, which is probably a critical event in triggering neuronal death when cellular acetylation homeostasis is impaired.

  14. Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

    SciTech Connect

    Hong, Wei, E-mail: hongwei@tijmu.edu.cn; College of Basic Medicine, Tianjin Medical University, 300070 Tianjin; Li, Jinru

    Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by whichmore » Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.« less

  15. A new strategy in the treatment of chemoresistant lung adenocarcinoma via specific siRNA transfection of SRF, E2F1, Survivin, HIF and STAT3.

    PubMed

    Stoleriu, Mircea Gabriel; Steger, Volker; Mustafi, Migdat; Michaelis, Martin; Cinatl, Jindrich; Schneider, Wilke; Nolte, Andrea; Kurz, Julia; Wendel, Hans Peter; Schlensak, Christian; Walker, Tobias

    2014-11-01

    According to the actual treatment strategies of lung cancer, the current therapeutic regimen is an individualized, multidisciplinary concept. The development of chemoresistance in the last decade represents the most important obstacle to an effective treatment. In our study, we examined a new therapeutic alternative in the treatment of multiresistant lung adenocarcinoma via siRNA-specific transfection of six crucial molecules involved in lung carcinogenesis [serum response factor(SFR), E2F1, Survivin, hypoxia inducible factor1 (HIF1), HIF2 and signal transducer and activator of transcription (STAT3)]. Three chemoresistant A549 adenocarcinoma cells were cultured under standard conditions at 37°C and 5% CO2. The chemoresistance against Vinflunine, Vinorelbine and Methotrexate was induced artificially. The A549 cells were transfected for 2 h at 37°C with specific siRNA targeting SRF, E2F1, Survivin, HIF1, HIF2 and STAT3 in a non-viral manner. The efficiency of siRNA silencing was evaluated via quantitative real-time polymerase chain reaction, whereas the surviving cells after siRNA transfection as predictor factor for tumoural growth were analysed with a CASY cell counter 3 days after transfection. The response of the chemotherapeutic resistant adenocarcinoma cells after siRNA transfection was concentration-dependent at both 25 and 100 nM. The CASY analysis showed a very effective suppression of adenocarcinoma cells in Vinorelbine, Vinflunine and Methotrexate groups, with significantly better results in comparison with the control group. In our study, we emphasized that siRNA interference might represent a productive platform for further research in order to investigate whether a new regimen in the treatment of multiresistant non-small-cell lung cancer could be established in vivo in the context of a multimodal cancer therapy. © The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights

  16. E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

    PubMed

    Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

    2017-10-02

    Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

  17. A lentiviral vector with expression controlled by E2F-1: A potential tool for the study and treatment of proliferative diseases

    SciTech Connect

    Strauss, Bryan E.; Patricio, Juliana Rotelli; Program in Biotechnology, University of Sao Paulo

    2006-10-06

    We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for thismore » factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.« less

  18. lncRNA-HIT promotes cell proliferation of non-small cell lung cancer by association with E2F1.

    PubMed

    Yu, L; Fang, F; Lu, S; Li, X; Yang, Y; Wang, Z

    2017-05-01

    Lung cancer is the leading cause of cancer-related death around the world. Long noncoding RNA (lncRNA) has pivotal roles in cancer occurrence and development. However, only a few lncRNAs have been functionally characterized. In the present study, we investigated the effects of lncRNA-HIT (HOXA transcript induced by TGFβ) expression on non-small cell lung cancer (NSCLC) cell phenotype with the gain-of-function and loss-of-function assays. We found that ectopic expression or knockdown of lncRNA-HIT markedly increased or decreased NSCLC cell proliferation, respectively. Moreover, we also showed that lncRNA-HIT interacted with E2F1 to regulate its target genes, such as Survivin, FOXM1, SKP2, NELL2 and DOK1. Collectively, our findings indicated that lncRNA-HIT affected the proliferation of NSCLC cells at least in part via regulating the occupancy of E2F1 in the promoter regions of its target genes. The lncRNA-HIT-E2F1 complex may be a potential target for NSCLC treatment.

  19. GTSE1: a novel TEAD4-E2F1 target gene involved in cell protrusions formation in triple-negative breast cancer cell models

    PubMed Central

    Stelitano, Debora; Leticia, Yamila Peche; Dalla, Emiliano; Monte, Martin; Piazza, Silvano; Schneider, Claudio

    2017-01-01

    GTSE1 over-expression has been reported as a potential marker for metastasis in various types of malignancies, including breast cancer. Despite this, the transcriptional regulation of this protein and the causes of its misregulation in tumors remain largely unknown. The aims of this work were to elucidate how GTSE1 is regulated at the transcriptional level and to clarify the mechanism underlying GTSE1-dependent cell functions in triple-negative breast cancer (TNBC). Here, we identified GTSE1 as a novel target gene of the TEAD4 transcription factor, highlighting a role for the YAP and TAZ coactivators in the transcriptional regulation of GTSE1. Moreover, we found that TEAD4 controls the formation of cell protrusions required for cell migration through GTSE1, unveiling a relevant effector role for this protein in the TEAD-dependent cellular functions and confirming TEAD4 role in promoting invasion and metastasis in breast cancer. Finally, we highlighted a role for the pRb-E2F1 pathway in the control of GTSE1 transcription and observed that treatment with drugs targeting the pRb-E2F1 or YAP/TAZ-TEAD pathways dramatically downregulated the expression levels of GTSE1 and of other genes involved in the formation of metastasis, suggesting their potential use in the treatment of TNBC. PMID:28978043

  20. Hypoxia inducible factor-1α regulates autophagy via the p27-E2F1 signaling pathway

    PubMed Central

    Wang, Pan; Long, Meijing; Zhang, Shijie; Cheng, Zhenyun; Zhao, Xin; He, Fucheng; Liu, Hongchun; Ming, Liang

    2017-01-01

    Autophagy is a highly conserved process by which the cell contents are delivered to lysosomes for degradation, or are used to provide macromolecules for energy generation under conditions of nutritional starvation. It has previously been demonstrated that cancer cells in hypoxic regions, with an oxygen concentration below the normal physiological level, express hypoxia inducible factor (HIF)-1α, in order to adapt and survive. HIF-1α is important in the regulation of oxygen homeostasis and the transcription of hundreds of genes in response to conditions of hypoxia, hence maintaining energy and redox homeostasis. To determine if HIF-1α modulates autophagy and the underlying molecular mechanisms regulating this process, the human esophageal cancer EC109 and IMR90 human diploid fibroblast cell lines were exposed to normoxic or hypoxic conditions and the expression levels of various proteins subsequently examined. Small interfering RNA was used to silence p27, in order to investigate its role in the process of HIF-1α regulated autophagy. Hypoxia induced autophagy in IMR90 cells and it was revealed that immature IMR90 cells demonstrated an increased rate of autophagy compared with mature cells. HIF-1α promoted EC109 cell autophagy via positively modulating p27, whereas silencing of p27 abolished the autophagy induced by hypoxia. The present study identified the primary components of the p27-E2F1 signaling pathway by which HIF-1α regulates autophagy. A previously unidentified mechanism is here presented, via which cancer cells may generate energy, or obtain macromolecules for survival. PMID:28627618

  1. Repression of miR-17-5p with elevated expression of E2F-1 and c-MYC in non-metastatic hepatocellular carcinoma and enhancement of cell growth upon reversing this expression pattern

    SciTech Connect

    El Tayebi, H.M.; Omar, K.; Hegy, S.

    2013-05-10

    Highlights: •The oncogenic miR-17-5p is downregulated in non-metastatic hepatocellular carcinoma patients. •E2F-1 and c-MYC transcripts are upregulated in non-metastatic HCC patients. •miR-17-5p forced overexpression inhibited E2F-1 and c-MYC expression in HuH-7 cells. •miR-17-5p mimicking increased HuH-7 cell growth, proliferation, migration and colony formation. •miR-17-5p is responsible for HCC progression among the c-MYC/E2F-1/miR-17-5p triad members. -- Abstract: E2F-1, c-MYC, and miR-17-5p is a triad of two regulatory loops: a negative and a positive loop, where c-MYC induces the expression of E2F-1 that induces the expression of miR-17-5p which in turn reverses the expression of E2F-1 to close the loop. In thismore » study, we investigated this triad for the first time in hepatocellular carcinoma (HCC), where miR-17-5p showed a significant down-regulation in 23 non-metastatic HCC biopsies compared to 10 healthy tissues; however, E2F-1 and c-MYC transcripts were markedly elevated. Forced over-expression of miR-17-5p in HuH-7 cells resulted in enhanced cell proliferation, growth, migration and clonogenicity with concomitant inhibition of E2F-1 and c-MYC transcripts expressions, while antagomirs of miR-17-5p reversed these events. In conclusion, this study revealed a unique pattern of expression for miR-17-5p in non-metastatic HCC patients in contrast to metastatic HCC patients. In addition we show that miR-17-5p is the key player among the triad that tumor growth and spread.« less

  2. Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes.

    PubMed

    Endorf, Elizabeth B; Qing, Hua; Aono, Jun; Terami, Naoto; Doyon, Geneviève; Hyzny, Eric; Jones, Karrie L; Findeisen, Hannes M; Bruemmer, Dennis

    2017-02-01

    Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC. © 2016 American Heart Association, Inc.

  3. HTLV-1 bZIP factor protein targets the Rb/E2F-1 pathway to promote proliferation and apoptosis of primary CD4+ T cells

    PubMed Central

    Kawatsuki, A; Yasunaga, J-i; Mitobe, Y; Green, PL; Matsuoka, M

    2016-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that induces a fatal T-cell malignancy, adult T-cell leukemia (ATL). Among several regulatory/accessory genes in HTLV-1, HTLV-1 bZIP factor (HBZ) is the only viral gene constitutively expressed in infected cells. Our previous study showed that HBZ functions in two different molecular forms, HBZ protein and HBZ RNA. In this study, we show that HBZ protein targets retinoblastoma protein (Rb), which is a critical tumor suppressor in many types of cancers. HBZ protein interacts with the Rb/E2F-1 complex and activates the transcription of E2F-target genes associated with cell cycle progression and apoptosis. Mouse primary CD4+ T cells transduced with HBZ show accelerated G1/S transition and apoptosis, and importantly, T cells from HBZ transgenic (HBZ-Tg) mice also demonstrate enhanced cell proliferation and apoptosis. To evaluate the functions of HBZ protein alone in vivo, we generated a new transgenic mouse strain that expresses HBZ mRNA altered by silent mutations but encoding intact protein. In these mice, the numbers of effector/memory and Foxp3+ T cells were increased, and genes associated with proliferation and apoptosis were upregulated. This study shows that HBZ protein promotes cell proliferation and apoptosis in primary CD4+ T cells through activation of the Rb/E2F pathway, and that HBZ protein also confers onto CD4+ T-cell immunophenotype similar to those of ATL cells, suggesting that HBZ protein has important roles in dysregulation of CD4+ T cells infected with HTLV-1. PMID:26804169

  4. Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.

    PubMed

    Leng, Feng; Yu, Jiekai; Zhang, Chunxiao; Alejo, Salvador; Hoang, Nam; Sun, Hong; Lu, Fei; Zhang, Hui

    2018-04-24

    Many non-histone proteins are lysine methylated and a novel function of this modification is to trigger the proteolysis of methylated proteins. Here, we report that the methylated lysine 142 of DNMT1, a major DNA methyltransferase that preserves epigenetic inheritance of DNA methylation patterns during DNA replication, is demethylated by LSD1. A novel methyl-binding protein, L3MBTL3, binds the K142-methylated DNMT1 and recruits a novel CRL4 DCAF5 ubiquitin ligase to degrade DNMT1. Both LSD1 and PHF20L1 act primarily in S phase to prevent DNMT1 degradation by L3MBTL3-CRL4 DCAF5 . Mouse L3MBTL3/MBT-1 deletion causes accumulation of DNMT1 protein, increased genomic DNA methylation, and late embryonic lethality. DNMT1 contains a consensus methylation motif shared by many non-histone proteins including E2F1, a key transcription factor for S phase. We show that the methylation-dependent E2F1 degradation is also controlled by L3MBTL3-CRL4 DCAF5 . Our studies elucidate for the first time a novel mechanism by which the stability of many methylated non-histone proteins are regulated.

  5. Nucleoprotein of influenza A virus negatively impacts antiapoptotic protein API5 to enhance E2F1-dependent apoptosis and virus replication.

    PubMed

    Mayank, A K; Sharma, S; Nailwal, H; Lal, S K

    2015-12-17

    Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.

  6. The Role of a Novel Nucleolar Protein in Regulation of E2F1 in Breast Cancer

    DTIC Science & Technology

    2009-09-01

    publication and successful defense of a PhD. 8 References 1. Paik JC, Wang B, Liu K, Lue J , Lin WC. Regulation of E2F1-induced apoptosis by...the nucleolar protein RRP1B. J Biol Chem. 2009 Dec 29. [E-pub ahead of print] 2. Hsieh SM, Look MP, Sieuwerts AM, Foekens JA, Hunter KW. Distinct...factor. J Biol Chem. 2009 Oct 16;284(42):28660-73. 4. Crawford NP, Walker RC, Lukes L, Officewala JS, Williams RW, Hunter KW. The Diasporin Pathway: a

  7. Mitochondrial ribosomal protein S18-2 is highly expressed in endometrial cancers along with free E2F1

    PubMed Central

    Iurchenko, Natalia; Kovalevska, Larysa; Stip, Maria C; Budnikova, Daria; Andersson, Sonia; Polischuk, Ludmila; Buchynska, Lubov; Kashuba, Elena

    2016-01-01

    Endometrial cancer (EC) is one of the most frequent causes of cancer death among women in developed countries. Histopathological diagnosis and imaging techniques for EC are limited, thus new prognostic markers are needed to offer patients the best treatment and follow-up. In the present paper we showed that the level of mitochondrial ribosomal protein MRPS18-2 (S18-2) increased in EC compared with the normal endometrium and hyperplasia, based on a study of 42 patient biopsies. Importantly, high expression of free E2F1 in EC correlates well with high S18-2 expression. The EC cell line HEC-1-A, which overexpresses S18-2 constitutively, showed an increased proliferation capacity in vitro and in vivo (in SCID mice). Moreover, pan-keratin, beta-catenin and E-cadherin signals are diminished in these cells, compared to the parental HEC-1-A line, in contrast to vimentin signal that is increased. This may be associated with epithelial-mesenchymal cell transition (EMT). We conclude that high expression of S18-2 and free E2F1, and low pan-keratin, beta-catenin, and E-cadherin signals might be a good set of prognostic markers for EC. PMID:26959119

  8. Fine-scale mapping of the FGFR2 breast cancer risk locus: putative functional variants differentially bind FOXA1 and E2F1.

    PubMed

    Meyer, Kerstin B; O'Reilly, Martin; Michailidou, Kyriaki; Carlebur, Saskia; Edwards, Stacey L; French, Juliet D; Prathalingham, Radhika; Dennis, Joe; Bolla, Manjeet K; Wang, Qin; de Santiago, Ines; Hopper, John L; Tsimiklis, Helen; Apicella, Carmel; Southey, Melissa C; Schmidt, Marjanka K; Broeks, Annegien; Van 't Veer, Laura J; Hogervorst, Frans B; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A; Lux, Michael P; Ekici, Arif B; Beckmann, Matthias W; Peto, Julian; Dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor J; Tomlinson, Ian; Kerin, Michael J; Miller, Nicola; Marme, Federick; Schneeweiss, Andreas; Sohn, Christof; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Menegaux, Florence; Bojesen, Stig E; Nordestgaard, Børge G; Nielsen, Sune F; Flyger, Henrik; Milne, Roger L; Zamora, M Pilar; Arias, Jose I; Benitez, Javier; Neuhausen, Susan; Anton-Culver, Hoda; Ziogas, Argyrios; Dur, Christina C; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Schmutzler, Rita K; Engel, Christoph; Ditsch, Nina; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Nevanlinna, Heli; Muranen, Taru A; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Yatabe, Yasushi; Dörk, Thilo; Helbig, Sonja; Bogdanova, Natalia V; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M; Chenevix-Trench, Georgia; Wu, Anna H; Tseng, Chiu-Chen; Van Den Berg, David; Stram, Daniel O; Lambrechts, Diether; Thienpont, Bernard; Christiaens, Marie-Rose; Smeets, Ann; Chang-Claude, Jenny; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Bonanni, Bernardo; Bernard, Loris; Couch, Fergus J; Olson, Janet E; Wang, Xianshu; Purrington, Kristen; Giles, Graham G; Severi, Gianluca; Baglietto, Laura; McLean, Catriona; Haiman, Christopher A; Henderson, Brian E; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S; Labrèche, France; Dumont, Martine; Teo, Soo-Hwang; Yip, Cheng-Har; Phuah, Sze-Yee; Kristensen, Vessela; Grenaker Alnæs, Grethe; Børresen-Dale, Anne-Lise; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Kauppila, Saila; Andrulis, Irene L; Knight, Julia A; Glendon, Gord; Tchatchou, Sandrine; Devilee, Peter; Tollenaar, Robert A E M; Seynaeve, Caroline M; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J; Lissowska, Jolanta; Czene, Kamila; Darabi, Hartef; Eriksson, Kimael; Hooning, Maartje J; Martens, John W M; van den Ouweland, Ans M W; van Deurzen, Carolien H M; Hall, Per; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Reed, Malcolm W R; Blot, William; Signorello, Lisa B; Cai, Qiuyin; Pharoah, Paul D P; Ghoussaini, Maya; Harrington, Patricia; Tyrer, Jonathan; Kang, Daehee; Choi, Ji-Yeob; Park, Sue K; Noh, Dong-Young; Hartman, Mikael; Hui, Miao; Lim, Wei-Yen; Buhari, Shaik A; Hamann, Ute; Försti, Asta; Rüdiger, Thomas; Ulmer, Hans-Ulrich; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Vachon, Celine; Slager, Susan; Fostira, Florentia; Pilarski, Robert; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Hou, Ming-Feng; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Schoemaker, Minouk J; Ponder, Bruce A J; Dunning, Alison M; Easton, Douglas F

    2013-12-05

    The 10q26 locus in the second intron of FGFR2 is the locus most strongly associated with estrogen-receptor-positive breast cancer in genome-wide association studies. We conducted fine-scale mapping in case-control studies genotyped with a custom chip (iCOGS), comprising 41 studies (n = 89,050) of European ancestry, 9 Asian ancestry studies (n = 13,983), and 2 African ancestry studies (n = 2,028) from the Breast Cancer Association Consortium. We identified three statistically independent risk signals within the locus. Within risk signals 1 and 3, genetic analysis identified five and two variants, respectively, highly correlated with the most strongly associated SNPs. By using a combination of genetic fine mapping, data on DNase hypersensitivity, and electrophoretic mobility shift assays to study protein-DNA binding, we identified rs35054928, rs2981578, and rs45631563 as putative functional SNPs. Chromatin immunoprecipitation showed that FOXA1 preferentially bound to the risk-associated allele (C) of rs2981578 and was able to recruit ERα to this site in an allele-specific manner, whereas E2F1 preferentially bound the risk variant of rs35054928. The risk alleles were preferentially found in open chromatin and bound by Ser5 phosphorylated RNA polymerase II, suggesting that the risk alleles are associated with changes in transcription. Chromatin conformation capture demonstrated that the risk region was able to interact with the promoter of FGFR2, the likely target gene of this risk region. A role for FOXA1 in mediating breast cancer susceptibility at this locus is consistent with the finding that the FGFR2 risk locus primarily predisposes to estrogen-receptor-positive disease. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  9. Fine-Scale Mapping of the FGFR2 Breast Cancer Risk Locus: Putative Functional Variants Differentially Bind FOXA1 and E2F1

    PubMed Central

    Meyer, Kerstin B.; O’Reilly, Martin; Michailidou, Kyriaki; Carlebur, Saskia; Edwards, Stacey L.; French, Juliet D.; Prathalingham, Radhika; Dennis, Joe; Bolla, Manjeet K.; Wang, Qin; de Santiago, Ines; Hopper, John L.; Tsimiklis, Helen; Apicella, Carmel; Southey, Melissa C.; Schmidt, Marjanka K.; Broeks, Annegien; Van ’t Veer, Laura J.; Hogervorst, Frans B.; Muir, Kenneth; Lophatananon, Artitaya; Stewart-Brown, Sarah; Siriwanarangsan, Pornthep; Fasching, Peter A.; Lux, Michael P.; Ekici, Arif B.; Beckmann, Matthias W.; Peto, Julian; dos Santos Silva, Isabel; Fletcher, Olivia; Johnson, Nichola; Sawyer, Elinor J.; Tomlinson, Ian; Kerin, Michael J.; Miller, Nicola; Marme, Federick; Schneeweiss, Andreas; Sohn, Christof; Burwinkel, Barbara; Guénel, Pascal; Truong, Thérèse; Laurent-Puig, Pierre; Menegaux, Florence; Bojesen, Stig E.; Nordestgaard, Børge G.; Nielsen, Sune F.; Flyger, Henrik; Milne, Roger L.; Zamora, M. Pilar; Arias, Jose I.; Benitez, Javier; Neuhausen, Susan; Anton-Culver, Hoda; Ziogas, Argyrios; Dur, Christina C.; Brenner, Hermann; Müller, Heiko; Arndt, Volker; Stegmaier, Christa; Meindl, Alfons; Schmutzler, Rita K.; Engel, Christoph; Ditsch, Nina; Brauch, Hiltrud; Brüning, Thomas; Ko, Yon-Dschun; Nevanlinna, Heli; Muranen, Taru A.; Aittomäki, Kristiina; Blomqvist, Carl; Matsuo, Keitaro; Ito, Hidemi; Iwata, Hiroji; Yatabe, Yasushi; Dörk, Thilo; Helbig, Sonja; Bogdanova, Natalia V.; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Kosma, Veli-Matti; Hartikainen, Jaana M.; Chenevix-Trench, Georgia; Wu, Anna H.; Tseng, Chiu-chen; Van Den Berg, David; Stram, Daniel O.; Lambrechts, Diether; Thienpont, Bernard; Christiaens, Marie-Rose; Smeets, Ann; Chang-Claude, Jenny; Rudolph, Anja; Seibold, Petra; Flesch-Janys, Dieter; Radice, Paolo; Peterlongo, Paolo; Bonanni, Bernardo; Bernard, Loris; Couch, Fergus J.; Olson, Janet E.; Wang, Xianshu; Purrington, Kristen; Giles, Graham G.; Severi, Gianluca; Baglietto, Laura; McLean, Catriona; Haiman, Christopher A.; Henderson, Brian E.; Schumacher, Fredrick; Le Marchand, Loic; Simard, Jacques; Goldberg, Mark S.; Labrèche, France; Dumont, Martine; Teo, Soo-Hwang; Yip, Cheng-Har; Phuah, Sze-Yee; Kristensen, Vessela; Grenaker Alnæs, Grethe; Børresen-Dale, Anne-Lise; Zheng, Wei; Deming-Halverson, Sandra; Shrubsole, Martha; Long, Jirong; Winqvist, Robert; Pylkäs, Katri; Jukkola-Vuorinen, Arja; Kauppila, Saila; Andrulis, Irene L.; Knight, Julia A.; Glendon, Gord; Tchatchou, Sandrine; Devilee, Peter; Tollenaar, Robert A.E.M.; Seynaeve, Caroline M.; García-Closas, Montserrat; Figueroa, Jonine; Chanock, Stephen J.; Lissowska, Jolanta; Czene, Kamila; Darabi, Hartef; Eriksson, Kimael; Hooning, Maartje J.; Martens, John W.M.; van den Ouweland, Ans M.W.; van Deurzen, Carolien H.M.; Hall, Per; Li, Jingmei; Liu, Jianjun; Humphreys, Keith; Shu, Xiao-Ou; Lu, Wei; Gao, Yu-Tang; Cai, Hui; Cox, Angela; Reed, Malcolm W.R.; Blot, William; Signorello, Lisa B.; Cai, Qiuyin; Pharoah, Paul D.P.; Ghoussaini, Maya; Harrington, Patricia; Tyrer, Jonathan; Kang, Daehee; Choi, Ji-Yeob; Park, Sue K.; Noh, Dong-Young; Hartman, Mikael; Hui, Miao; Lim, Wei-Yen; Buhari, Shaik A.; Hamann, Ute; Försti, Asta; Rüdiger, Thomas; Ulmer, Hans-Ulrich; Jakubowska, Anna; Lubinski, Jan; Jaworska, Katarzyna; Durda, Katarzyna; Sangrajrang, Suleeporn; Gaborieau, Valerie; Brennan, Paul; McKay, James; Vachon, Celine; Slager, Susan; Fostira, Florentia; Pilarski, Robert; Shen, Chen-Yang; Hsiung, Chia-Ni; Wu, Pei-Ei; Hou, Ming-Feng; Swerdlow, Anthony; Ashworth, Alan; Orr, Nick; Schoemaker, Minouk J.; Ponder, Bruce A.J.; Dunning, Alison M.; Easton, Douglas F.

    2013-01-01

    The 10q26 locus in the second intron of FGFR2 is the locus most strongly associated with estrogen-receptor-positive breast cancer in genome-wide association studies. We conducted fine-scale mapping in case-control studies genotyped with a custom chip (iCOGS), comprising 41 studies (n = 89,050) of European ancestry, 9 Asian ancestry studies (n = 13,983), and 2 African ancestry studies (n = 2,028) from the Breast Cancer Association Consortium. We identified three statistically independent risk signals within the locus. Within risk signals 1 and 3, genetic analysis identified five and two variants, respectively, highly correlated with the most strongly associated SNPs. By using a combination of genetic fine mapping, data on DNase hypersensitivity, and electrophoretic mobility shift assays to study protein-DNA binding, we identified rs35054928, rs2981578, and rs45631563 as putative functional SNPs. Chromatin immunoprecipitation showed that FOXA1 preferentially bound to the risk-associated allele (C) of rs2981578 and was able to recruit ERα to this site in an allele-specific manner, whereas E2F1 preferentially bound the risk variant of rs35054928. The risk alleles were preferentially found in open chromatin and bound by Ser5 phosphorylated RNA polymerase II, suggesting that the risk alleles are associated with changes in transcription. Chromatin conformation capture demonstrated that the risk region was able to interact with the promoter of FGFR2, the likely target gene of this risk region. A role for FOXA1 in mediating breast cancer susceptibility at this locus is consistent with the finding that the FGFR2 risk locus primarily predisposes to estrogen-receptor-positive disease. PMID:24290378

  10. Transactivation of micrornA-320 by microRNA-383 regulates granulosa cell functions by targeting E2F1 and SF-1 proteins.

    PubMed

    Yin, Mianmian; Wang, Xiaorong; Yao, Guidong; Lü, Mingrong; Liang, Meng; Sun, Yingpu; Sun, Fei

    2014-06-27

    Our previous studies have shown that microRNA-320 (miR-320) is one of the most down-regulated microRNAs (miRNA) in mouse ovarian granulosa cells (GCs) after TGF-β1 treatment. However, the underlying mechanisms of miR-320 involved in GC function during follicular development remain unknown. In this study, we found that pregnant mare serum gonadotropin treatment resulted in the suppression of miR-320 expression in a time-dependent manner. miR-320 was mainly expressed in GCs and oocytes of mouse ovarian follicles in follicular development. Overexpression of miR-320 inhibited estradiol synthesis and proliferation of GCs through targeting E2F1 and SF-1. E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. FSH down-regulated the expression of miR-320 and regulated the function of miR-320 in mouse GCs. miR-383 promoted the expression of miR-320 and enhanced miR-320-mediated suppression of GC proliferation. Injection of miR-320 into the ovaries of mice partially promoted the production of testosterone and progesterone but inhibited estradiol release in vivo. Moreover, the expression of miR-320 and miR-383 was up-regulated in the follicular fluid of polycystic ovarian syndrome patients, although the expression of E2F1 and SF-1 was down-regulated in GCs. These data demonstrated that miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development. Understanding the regulation of miRNA biogenesis and function in the follicular development will potentiate the usefulness of miRNA in the treatment of reproduction and some steroid-related disorders. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The TERT promoter SNP rs2853669 decreases E2F1 transcription factor binding and increases mortality and recurrence risks in liver cancer.

    PubMed

    Ko, Eunkyong; Seo, Hyun-Wook; Jung, Eun Sun; Kim, Baek-hui; Jung, Guhung

    2016-01-05

    A common single-nucleotide polymorphism in the telomerase reverse transcriptase (TERT) promoter, rs2853669 influences patient survival rates and the risk of developing cancer. Recently, several lines of evidence suggest that the rs2853669 suppresses TERT promoter mutation-mediated TERT expression levels and cancer mortality as well as recurrence rates. However, no reports are available on the impact of rs2853669 on TERT expression in hepatocellular carcinoma (HCC) and its association with patient survival. Here, we found that HCC-related overall and recurrence-free survival rates were not associated with TERT promoter mutation individually, but rs2853669 and the TERT promoter mutation in combination were associated with poor survival rates. TERT mRNA expression and telomere fluorescence levels were greater in patients with HCC who had both the combination. The combination caused TERT promoter methylation through regulating the binding of DNA methyltransferase 1 and histone deacetylase 1 to the TERT promoter in HCC cell lines. The TERT expression level was significantly higher in HCC tumor with a methylated promoter than in that with an unmethylated promoter. In conclusion, we demonstrate a substantial role for the rs2853669 in HCC with TERT promoter mutation, which suggests that the combination of the rs2853669 and the mutation indicate poor prognoses in liver cancer.

  12. Comparative modeling and docking studies of p16ink4/cyclin D1/Rb pathway genes in lung cancer revealed functionally interactive residue of RB1 and its functional partner E2F1.

    PubMed

    Naqsh e Zahra, Syeda; Khattak, Naureen Aslam; Mir, Asif

    2013-01-01

    Lung cancer is the major cause of mortality worldwide. Major signalling pathways that could play significant role in lung cancer therapy include (1) Growth promoting pathways (Epidermal Growth Factor Receptor/Ras/ PhosphatidylInositol 3-Kinase) (2) Growth inhibitory pathways (p53/Rb/P14ARF, STK11) (3) Apoptotic pathways (Bcl-2/Bax/Fas/FasL). Insilico strategy was implemented to solve the mystery behind selected lung cancer pathway by applying comparative modeling and molecular docking studies. YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of -0.132 and -0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1. This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of candidate genes and their important interacting residues likely

  13. Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F.

    PubMed

    Oliveira, Amanda; Beyer, Georg; Chugh, Rohit; Skube, Steven J; Majumder, Kaustav; Banerjee, Sulagna; Sangwan, Veena; Li, Lihua; Dawra, Rajinder; Subramanian, Subbaya; Saluja, Ashok; Dudeja, Vikas

    2015-06-01

    Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1-S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude

  14. SUMOylation of DRIL1 Directs Its Transcriptional Activity Towards Leukocyte Lineage-Specific Genes

    PubMed Central

    van Lohuizen, Maarten; Peeper, Daniel S.

    2009-01-01

    DRIL1 is an ARID family transcription factor that can immortalize primary mouse fibroblasts, bypass RASV12-induced cellular senescence and collaborate with RASV12 or MYC in mediating oncogenic transformation. It also activates immunoglobulin heavy chain transcription and engages in heterodimer formation with E2F to stimulate E2F-dependent transcription. Little, however, is known about the regulation of DRIL1 activity. Recently, DRIL1 was found to interact with the SUMO-conjugating enzyme Ubc9, but the functional relevance of this association has not been assessed. Here, we show that DRIL1 is sumoylated both in vitro and in vivo at lysine 398. Moreover, we provide evidence that PIASy functions as a specific SUMO E3-ligase for DRIL1 and promotes its sumoylation both in vitro and in vivo. Furthermore, consistent with the subnuclear localization of PIASy in the Matrix-Associated Region (MAR), SUMO-modified DRIL1 species are found exclusively in the MAR fraction. This post-translational modification interferes neither with the subcellular localization nor the DNA-binding activity of the protein. In contrast, DRIL1 sumoylation impairs its interaction with E2F1 in vitro and modifies its transcriptional activity in vivo, driving transcription of subset of genes regulating leukocyte fate. Taken together, these results identify sumoylation as a novel post-translational modification of DRIL1 that represents an important mechanism for targeting and modulating DRIL1 transcriptional activity. PMID:19436740

  15. Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes.

    PubMed

    Zellmer, Sebastian; Schmidt-Heck, Wolfgang; Godoy, Patricio; Weng, Honglei; Meyer, Christoph; Lehmann, Thomas; Sparna, Titus; Schormann, Wiebke; Hammad, Seddik; Kreutz, Clemens; Timmer, Jens; von Weizsäcker, Fritz; Thürmann, Petra A; Merfort, Irmgard; Guthke, Reinhard; Dooley, Steven; Hengstler, Jan G; Gebhardt, Rolf

    2010-12-01

    The cellular basis of liver regeneration has been intensely investigated for many years. However, the mechanisms initiating hepatocyte "plasticity" and priming for proliferation are not yet fully clear. We investigated alterations in gene expression patterns during the first 72 hours of C57BL/6N mouse hepatocyte culture on collagen monolayers (CM), which display a high basal frequency of proliferation in the absence of cytokines. Although many metabolic genes were down-regulated, genes related to mitogen-activated protein kinase (MAPK) signaling and cell cycle were up-regulated. The latter genes showed an overrepresentation of transcription factor binding sites (TFBS) for ETF (TEA domain family member 2), E2F1 (E2F transcription factor 1), and SP-1 (Sp1 transcription factor) (P < 0.001), all depending on MAPK signaling. Time-dependent increase of ERK1/2 phosphorylation occurred during the first 48 hours (and beyond) in the absence of cytokines, accompanied by an enhanced bromodeoxyuridine labeling index of 20%. The MEK inhibitor PD98059 blunted these effects indicating MAPK signaling as major trigger for this cytokine-independent proliferative response. In line with these in vitro findings, liver tissue of mice challenged with CCl(4) displayed hepatocytes with intense p-ERK1/2 staining and nuclear SP-1 and E2F1 expression. Furthermore, differentially expressed genes in mice after partial hepatectomy contained overrepresented TFBS for ETF, E2F1, and SP-1 and displayed increased expression of E2F1. Cultivation of murine hepatocytes on CM primes cells for proliferation through cytokine-independent activation of MAPK signaling. The transcription factors ETF, E2F1, and SP-1 seem to play a pronounced role in mediating proliferation-dependent differential gene expression. Similar events, but on a shorter time-scale, occur very early after liver damage in vivo. Copyright © 2010 American Association for the Study of Liver Diseases.

  16. C3 exoenzyme impairs cell proliferation and apoptosis by altering the activity of transcription factors.

    PubMed

    von Elsner, Leonie; Hagemann, Sandra; Just, Ingo; Rohrbeck, Astrid

    2016-09-01

    C3 exoenzyme from C. botulinum is an ADP-ribosyltransferase that inactivates selectively RhoA, B, and C by coupling an ADP-ribose moiety. Rho-GTPases are involved in various cellular processes, such as regulation of actin cytoskeleton, cell proliferation, and apoptosis. Previous studies of our group with the murine hippocampal cell line HT22 revealed a C3-mediated inhibition of cell proliferation after 48 h and a prevention of serum-starved cells from apoptosis. For both effects, alterations of various signaling pathways are already known, including also changes on the transcriptional level. Investigations on the transcriptional activity in HT22 cells treated with C3 for 48 h identified five out of 48 transcription factors namely Sp1, ATF2, E2F-1, CBF, and Stat6 with a significantly regulated activity. For validation of identified transcription factors, studies on the protein level of certain target genes were performed. Western blot analyses exhibited an enhanced abundance of Sp1 target genes p21 and COX-2 as well as an increase in phosphorylation of c-Jun. In contrast, the level of p53 and apoptosis-inducing GADD153, a target gene of ATF2, was decreased. Our results reveal that C3 regulates the transcriptional activity of Sp1 and ATF2 resulting downstream in an altered protein abundance of various target genes. As the affected proteins are involved in the regulation of cell proliferation and apoptosis, thus the C3-mediated anti-proliferative and anti-apoptotic effects are consequences of the Rho-dependent alterations of the activity of certain transcriptional factors.

  17. MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.

    PubMed

    Christensen, Lise Lotte; Tobiasen, Heidi; Holm, Anja; Schepeler, Troels; Ostenfeld, Marie S; Thorsen, Kasper; Rasmussen, Mads H; Birkenkamp-Demtroeder, Karin; Sieber, Oliver M; Gibbs, Peter; Lubinski, Jan; Lamy, Philippe; Laurberg, Søren; Oster, Bodil; Hansen, Kristian Q; Hagemann-Madsen, Rikke; Byskov, Kristina; Ørntoft, Torben F; Andersen, Claus L

    2013-07-01

    Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1. Copyright © 2012 UICC.

  18. E2F transcription factor-1 deficiency reduces pathophysiology in the mouse model of Duchenne muscular dystrophy through increased muscle oxidative metabolism.

    PubMed

    Blanchet, Emilie; Annicotte, Jean-Sébastien; Pradelli, Ludivine A; Hugon, Gérald; Matecki, Stéfan; Mornet, Dominique; Rivier, François; Fajas, Lluis

    2012-09-01

    E2F1 deletion leads to increased mitochondrial number and function, increased body temperature in response to cold and increased resistance to fatigue with exercise. Since E2f1-/- mice show increased muscle performance, we examined the effect of E2f1 genetic inactivation in the mdx background, a mouse model of Duchenne muscular dystrophy (DMD). E2f1-/-;mdx mice demonstrated a strong reduction of physiopathological signs of DMD, including preservation of muscle structure, decreased inflammatory profile, increased utrophin expression, resulting in better endurance and muscle contractile parameters, comparable to normal mdx mice. E2f1 deficiency in the mdx genetic background increased the oxidative metabolic gene program, mitochondrial activity and improved muscle functions. Interestingly, we observed increased E2F1 protein levels in DMD patients, suggesting that E2F1 might represent a promising target for the treatment of DMD.

  19. Linking Smads and transcriptional activation.

    PubMed

    Inman, Gareth J

    2005-02-15

    TGF-beta1 (transforming growth factor-beta1) is the prototypical member of a large family of pleiotropic cytokines that regulate diverse biological processes during development and adult tissue homoeostasis. TGF-beta signals via membrane bound serine/threonine kinase receptors which transmit their signals via the intracellular signalling molecules Smad2, Smad3 and Smad4. These Smads contain conserved MH1 and MH2 domains separated by a flexible linker domain. Smad2 and Smad3 act as kinase substrates for the receptors, and, following phosphorylation, they form complexes with Smad4 and translocate to the nucleus. These Smad complexes regulate gene expression and ultimately determine the biological response to TGF-beta. In this issue of the Biochemical Journal, Wang et al. have shown that, like Smad4, the linker domain of Smad3 contains a Smad transcriptional activation domain. This is capable of recruiting the p300 transcriptional co-activator and is required for Smad3-dependent transcriptional activation. This study raises interesting questions about the nature and regulation of Smad-regulated gene activation and elevates the status of the linker domain to rival that of the much-lauded MH1 and MH2 domains.

  20. Network-based differential gene expression analysis suggests cell cycle related genes regulated by E2F1 underlie the molecular difference between smoker and non-smoker lung adenocarcinoma

    PubMed Central

    2013-01-01

    Background Differential gene expression (DGE) analysis is commonly used to reveal the deregulated molecular mechanisms of complex diseases. However, traditional DGE analysis (e.g., the t test or the rank sum test) tests each gene independently without considering interactions between them. Top-ranked differentially regulated genes prioritized by the analysis may not directly relate to the coherent molecular changes underlying complex diseases. Joint analyses of co-expression and DGE have been applied to reveal the deregulated molecular modules underlying complex diseases. Most of these methods consist of separate steps: first to identify gene-gene relationships under the studied phenotype then to integrate them with gene expression changes for prioritizing signature genes, or vice versa. It is warrant a method that can simultaneously consider gene-gene co-expression strength and corresponding expression level changes so that both types of information can be leveraged optimally. Results In this paper, we develop a gene module based method for differential gene expression analysis, named network-based differential gene expression (nDGE) analysis, a one-step integrative process for prioritizing deregulated genes and grouping them into gene modules. We demonstrate that nDGE outperforms existing methods in prioritizing deregulated genes and discovering deregulated gene modules using simulated data sets. When tested on a series of smoker and non-smoker lung adenocarcinoma data sets, we show that top differentially regulated genes identified by the rank sum test in different sets are not consistent while top ranked genes defined by nDGE in different data sets significantly overlap. nDGE results suggest that a differentially regulated gene module, which is enriched for cell cycle related genes and E2F1 targeted genes, plays a role in the molecular differences between smoker and non-smoker lung adenocarcinoma. Conclusions In this paper, we develop nDGE to prioritize

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

    PubMed

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Chromatin insulation by a transcriptional activator

    PubMed Central

    Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    2003-01-01

    In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

  3. Profilin is associated with transcriptionally active genes

    PubMed Central

    Söderberg, Emilia; Hessle, Viktoria; von Euler, Anne; Visa, Neus

    2012-01-01

    We have raised antibodies against the profilin of Chironomus tentans to study the location of profilin relative to chromatin and to active genes in salivary gland polytene chromosomes. We show that a fraction of profilin is located in the nucleus, where profilin is highly concentrated in the nucleoplasm and at the nuclear periphery. Moreover, profilin is associated with multiple bands in the polytene chromosomes. By staining salivary glands with propidium iodide, we show that profilin does not co-localize with dense chromatin. Profilin associates instead with protein-coding genes that are transcriptionally active, as revealed by co-localization with hnRNP and snRNP proteins. We have performed experiments of transcription inhibition with actinomycin D and we show that the association of profilin with the chromosomes requires ongoing transcription. However, the interaction of profilin with the gene loci does not depend on RNA. Our results are compatible with profilin regulating actin polymerization in the cell nucleus. However, the association of actin with the polytene chromosomes of C. tentans is sensitive to RNase, whereas the association of profilin is not, and we propose therefore that the chromosomal location of profilin is independent of actin. PMID:22572953

  4. Mechanisms of specificity in neuronal activity-regulated gene transcription

    PubMed Central

    Lyons, Michelle R.; West, Anne E.

    2011-01-01

    The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. PMID:21620929

  5. The Smad3 linker region contains a transcriptional activation domain

    PubMed Central

    2004-01-01

    Transforming growth factor-β (TGF-β)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-β/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-β transcriptional activation responses, although it can be phosphorylated by the TGF-β receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-β. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control. PMID:15588252

  6. The Smad3 linker region contains a transcriptional activation domain.

    PubMed

    Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

    2005-02-15

    Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.

  7. Transcription through enhancers suppresses their activity in Drosophila

    PubMed Central

    2013-01-01

    Background Enhancer elements determine the level of target gene transcription in a tissue-specific manner, providing for individual patterns of gene expression in different cells. Knowledge of the mechanisms controlling enhancer action is crucial for understanding global regulation of transcription. In particular, enhancers are often localized within transcribed regions of the genome. A number of experiments suggest that transcription can have both positive and negative effects on regulatory elements. In this study, we performed direct tests for the effect of transcription on enhancer activity. Results Using a transgenic reporter system, we investigated the relationship between the presence of pass-through transcription and the activity of Drosophila enhancers controlling the expression of the white and yellow genes. The results show that transcription from different promoters affects the activity of enhancers, counteracting their ability to activate the target genes. As expected, the presence of a transcriptional terminator between the inhibiting promoter and the affected enhancer strongly reduces the suppression. Moreover, transcription leads to dislodging of the Zeste protein that is responsible for the enhancer-dependent regulation of the white gene, suggesting a 'transcription interference’ mechanism for this regulation. Conclusions Our findings suggest a role for pass-through transcription in negative regulation of enhancer activity. PMID:24279291

  8. Asymmetry between Activation and Deactivation during a Transcriptional Pulse.

    PubMed

    Dunham, Lee S S; Momiji, Hiroshi; Harper, Claire V; Downton, Polly J; Hey, Kirsty; McNamara, Anne; Featherstone, Karen; Spiller, David G; Rand, David A; Finkenstädt, Bärbel; White, Michael R H; Davis, Julian R E

    2017-12-27

    Transcription in eukaryotic cells occurs in gene-specific bursts or pulses of activity. Recent studies identified a spectrum of transcriptionally active "on-states," interspersed with periods of inactivity, but these "off-states" and the process of transcriptional deactivation are poorly understood. To examine what occurs during deactivation, we investigate the dynamics of switching between variable rates. We measured live single-cell expression of luciferase reporters from human growth hormone or human prolactin promoters in a pituitary cell line. Subsequently, we applied a statistical variable-rate model of transcription, validated by single-molecule FISH, to estimate switching between transcriptional rates. Under the assumption that transcription can switch to any rate at any time, we found that transcriptional activation occurs predominantly as a single switch, whereas deactivation occurs with graded, stepwise decreases in transcription rate. Experimentally altering cAMP signalling with forskolin or chromatin remodelling with histone deacetylase inhibitor modifies the duration of defined transcriptional states. Our findings reveal transcriptional activation and deactivation as mechanistically independent, asymmetrical processes. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. From DNA binding to transcriptional activation: Is the TALE complete?

    PubMed

    Bobola, Nicoletta

    2017-09-04

    How transcription factors (TFs) control enhancer and promoter functions to effect changes in gene expression is an important question. In this issue, Hau et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201701154) show that the TALE TF MEIS recruits the histone modifier PARP1/ARTD1 at promoters to decompact chromatin and activate transcription. © 2017 Bobola.

  10. Elucidation of Small RNAs that Activate Transcription in Bacteria

    DTIC Science & Technology

    2012-03-01

    bacterial sRNAs that activate transcription of a target gene in E. coli to varying degrees. Mutation of the strongest activator modified its...identified RNA- based transcriptional activators in yeast (Buskirk et al., 2003) although the underlying mechanism was not elucidated. We show that the...previous yeast two-hybrid (Buskirk et al., 2003) and three-hybrid studies (Bernstein et al., 2002). Colonies were observed from co-transformations of pBT

  11. Selective Activation of Transcription by a Novel CCAAT Binding Factor

    NASA Astrophysics Data System (ADS)

    Maity, Sankar N.; Golumbek, Paul T.; Karsenty, Gerard; de Crombrugghe, Benoit

    1988-07-01

    A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the α 2(I) collagen, the α 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

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

    PubMed

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    PubMed

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Regulation of transcriptional activators by DNA-binding domain ubiquitination

    PubMed Central

    Landré, Vivien; Revi, Bhindu; Mir, Maria Gil; Verma, Chandra; Hupp, Ted R; Gilbert, Nick; Ball, Kathryn L

    2017-01-01

    Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation. PMID:28362432

  15. HTLV-1 Tax activates HIV-1 transcription in latency models.

    PubMed

    Geddes, Victor Emmanuel Viana; José, Diego Pandeló; Leal, Fabio E; Nixon, Douglas F; Tanuri, Amilcar; Aguiar, Renato Santana

    2017-04-01

    HIV-1 latency is a major obstacle to HIV-1 eradication. Coinfection with HTLV-1 has been associated with faster progression to AIDS. HTLV-1 encodes the transactivator Tax which can activate both HTLV-1 and HIV-1 transcription. Here, we demonstrate that Tax activates HIV transcription in latent CD4 + T cells. Tax promotes the activation of P-TEFb, releasing CDK9 and Cyclin T1 from inactive forms, promoting transcription elongation and reactivation of latent HIV-1. Tax mutants lacking interaction with the HIV-1-LTR promoter were not able to activate P-TEFb, with no subsequent activation of latent HIV. In HIV-infected primary resting CD4 + T cells, Tax-1 reactivated HIV-1 transcription up to five fold, confirming these findings in an ex vivo latency model. Finally, our results confirms that HTLV-1/Tax hijacks cellular partners, promoting HIV-1 transcription, and this interaction should be further investigated in HIV-1 latency studies in patients with HIV/HTLV-1 co-infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Transcription Factor Activities Enhance Markers of Drug Sensitivity in Cancer.

    PubMed

    Garcia-Alonso, Luz; Iorio, Francesco; Matchan, Angela; Fonseca, Nuno; Jaaks, Patricia; Peat, Gareth; Pignatelli, Miguel; Falcone, Fiammetta; Benes, Cyril H; Dunham, Ian; Bignell, Graham; McDade, Simon S; Garnett, Mathew J; Saez-Rodriguez, Julio

    2018-02-01

    Transcriptional dysregulation induced by aberrant transcription factors (TF) is a key feature of cancer, but its global influence on drug sensitivity has not been examined. Here, we infer the transcriptional activity of 127 TFs through analysis of RNA-seq gene expression data newly generated for 448 cancer cell lines, combined with publicly available datasets to survey a total of 1,056 cancer cell lines and 9,250 primary tumors. Predicted TF activities are supported by their agreement with independent shRNA essentiality profiles and homozygous gene deletions, and recapitulate mutant-specific mechanisms of transcriptional dysregulation in cancer. By analyzing cell line responses to 265 compounds, we uncovered numerous TFs whose activity interacts with anticancer drugs. Importantly, combining existing pharmacogenomic markers with TF activities often improves the stratification of cell lines in response to drug treatment. Our results, which can be queried freely at dorothea.opentargets.io, offer a broad foundation for discovering opportunities to refine personalized cancer therapies. Significance: Systematic analysis of transcriptional dysregulation in cancer cell lines and patient tumor specimens offers a publicly searchable foundation to discover new opportunities to refine personalized cancer therapies. Cancer Res; 78(3); 769-80. ©2017 AACR . ©2017 American Association for Cancer Research.

  17. Identifying transcription factor functions and targets by phenotypic activation

    PubMed Central

    Chua, Gordon; Morris, Quaid D.; Sopko, Richelle; Robinson, Mark D.; Ryan, Owen; Chan, Esther T.; Frey, Brendan J.; Andrews, Brenda J.; Boone, Charles; Hughes, Timothy R.

    2006-01-01

    Mapping transcriptional regulatory networks is difficult because many transcription factors (TFs) are activated only under specific conditions. We describe a generic strategy for identifying genes and pathways induced by individual TFs that does not require knowledge of their normal activation cues. Microarray analysis of 55 yeast TFs that caused a growth phenotype when overexpressed showed that the majority caused increased transcript levels of genes in specific physiological categories, suggesting a mechanism for growth inhibition. Induced genes typically included established targets and genes with consensus promoter motifs, if known, indicating that these data are useful for identifying potential new target genes and binding sites. We identified the sequence 5′-TCACGCAA as a binding sequence for Hms1p, a TF that positively regulates pseudohyphal growth and previously had no known motif. The general strategy outlined here presents a straightforward approach to discovery of TF activities and mapping targets that could be adapted to any organism with transgenic technology. PMID:16880382

  18. Arsenic-induced Aurora-A activation contributes to chromosome instability and tumorigenesis

    NASA Astrophysics Data System (ADS)

    Wu, Chin-Han; Tseng, Ya-Shih; Yang, Chao-Chun; Kao, Yu-Ting; Sheu, Hamm-Ming; Liu, Hsiao-Sheng

    2013-11-01

    Arsenic may cause serious environmental pollution and is a serious industrial problem. Depending on the dosage, arsenic may trigger the cells undergoing either proliferation or apoptosis-related cell death. Because of lack of the proper animal model to study arsenic induced tumorigenesis, the accurate risk level of arsenic exposure has not been determined. Arsenic shows genotoxic effect on human beings who uptake water contaminated by arsenic. Chromosome aberration is frequently detected in arsenic exposure-related diseases and is associated with increased oxidative stress and decreased DNA repairing activity, but the underlying mechanism remains elusive. Aurora-A is a mitotic kinase, over-expression of Aurora-A leads to centrosome amplification, chromosomal instability and cell transformation. We revealed that Aurora-A is over-expressed in the skin and bladder cancer patients from blackfoot-disease endemic areas. Our cell line studies reveal that arsenic exposure between 0.5 μM and 1 μM for 2-7 days are able to induce Aurora-A expression and activation based on promoter activity, RNA and protein analysis. Aurora-A overexpression further increases the frequency of unsymmetrical chromosome segregation through centrosome amplification followed by cell population accumulated at S phase in immortalized keratinocyte (HaCaT) and uroepithelial cells (E7). Furthermore, Aurora-A over-expression was sustained for 1-4 weeks by chronic treatment of immortalized bladder and skin cells with NaAsO2. Aurora-A promoter methylation and gene amplification was not detected in the long-term arsenic treated E7 cells. Furthermore, the expression level of E2F1 transcription factor (E2F1) is increased in the presence of arsenic, and arsenic-related Aurora-A over-expression is transcriptionally regulated by E2F1. We further demonstrated that overexpression of Aurora-A and mutant Ha-ras or Aurora-A and mutant p53 may act additively to trigger arsenic-related bladder and skin cancer

  19. Transcriptional activity of transposable elements in coelacanth.

    PubMed

    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. © 2013 Wiley Periodicals, Inc.

  20. Novel Transcriptional Activities of Vitamin E: Inhibition of Cholesterol Biosynthesis

    PubMed Central

    Valastyan, Scott; Thakur, Varsha; Johnson, Amy; Kumar, Karan; Manor, Danny

    2008-01-01

    Vitamin E is a dietary lipid that is essential for vertebrate health and fertility. The biological activity of vitamin E is thought to reflect its ability to quench oxygen- and carbon- based free radicals, and thus to protect the organism from oxidative damage. However, recent reports suggest that vitamin E may also display other biological activities. Here, to examine possible mechanisms that may underlie such non-classical activities of vitamin E, we investigated the possibility that it functions as a specific modulator of gene expression. We show that treatment of cultured hepatocytes with RRR-α-tocopherol alters the expression of multiple genes and that these effects are distinct from those elicited by another antioxidant. Genes modulated by vitamin E include those that encode key enzymes in the cholesterol biosynthetic pathway. Correspondingly, vitamin E caused a pronounced inhibition of de novo cholesterol biosynthesis. The transcriptional activities of vitamin E were mediated by attenuating the post-translational processing of the transcription factor SREBP-2 that, in turn, led to a decreased transcriptional activity of sterol responsive elements in the promoters of target genes. These observations indicate that vitamin E possesses novel transcriptional activities that affect fundamental biological processes. Cross talk between tocopherol levels and cholesterol status may be an important facet of the biological activities of vitamin E. PMID:18095660

  1. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  2. Multivalency regulates activity in an intrinsically disordered transcription factor

    PubMed Central

    Clark, Sarah; Myers, Janette B; King, Ashleigh; Fiala, Radovan; Novacek, Jiri; Pearce, Grant; Heierhorst, Jörg; Reichow, Steve L

    2018-01-01

    The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference for low occupancy complexes at saturating LC8 concentrations with both human and Drosophila ASCIZ indicates that negative cooperativity is an important feature of ASCIZ-LC8 interactions. The prevalence of intrinsic disorder and multivalency among transcription factors suggests that formation of heterogeneous, dynamic complexes is a widespread mechanism for tuning transcriptional regulation. PMID:29714690

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

    PubMed Central

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

    2015-01-01

    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. PMID:25588787

  4. Aerobic glycolysis tunes YAP/TAZ transcriptional activity

    PubMed Central

    Enzo, Elena; Santinon, Giulia; Pocaterra, Arianna; Aragona, Mariaceleste; Bresolin, Silvia; Forcato, Mattia; Grifoni, Daniela; Pession, Annalisa; Zanconato, Francesca; Guzzo, Giulia; Bicciato, Silvio; Dupont, Sirio

    2015-01-01

    Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro-tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth-promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro-tumorigenic activities such as YAP/TAZ. PMID:25796446

  5. Polycomb repressive complex 1 modifies transcription of active genes

    PubMed Central

    Pherson, Michelle; Misulovin, Ziva; Gause, Maria; Mihindukulasuriya, Kathie; Swain, Amanda; Dorsett, Dale

    2017-01-01

    This study examines the role of Polycomb repressive complex 1 (PRC1) at active genes. The PRC1 and PRC2 complexes are crucial for epigenetic silencing during development of an organism. They are recruited to Polycomb response elements (PREs) and establish silenced domains over several kilobases. Recent studies show that PRC1 is also directly recruited to active genes by the cohesin complex. Cohesin participates broadly in control of gene transcription, but it is unknown whether cohesin-recruited PRC1 also plays a role in transcriptional control of active genes. We address this question using genome-wide RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). The results show that PRC1 influences transcription of active genes, and a significant fraction of its effects are likely direct. The roles of different PRC1 subunits can also vary depending on the gene. Depletion of PRC1 subunits by RNA interference alters phosphorylation of RNA polymerase II (Pol II) and occupancy by the Spt5 pausing-elongation factor at most active genes. These effects on Pol II phosphorylation and Spt5 are likely linked to changes in elongation and RNA processing detected by nascent RNA-seq, although the mechanisms remain unresolved. The experiments also reveal that PRC1 facilitates association of Spt5 with enhancers and PREs. Reduced Spt5 levels at these regulatory sequences upon PRC1 depletion coincide with changes in Pol II occupancy and phosphorylation. Our findings indicate that, in addition to its repressive roles in epigenetic gene silencing, PRC1 broadly influences transcription of active genes and may suppress transcription of nonpromoter regulatory sequences. PMID:28782042

  6. Combinatorial influence of environmental parameters on transcription factor activity.

    PubMed

    Knijnenburg, T A; Wessels, L F A; Reinders, M J T

    2008-07-01

    Cells receive a wide variety of environmental signals, which are often processed combinatorially to generate specific genetic responses. Changes in transcript levels, as observed across different environmental conditions, can, to a large extent, be attributed to changes in the activity of transcription factors (TFs). However, in unraveling these transcription regulation networks, the actual environmental signals are often not incorporated into the model, simply because they have not been measured. The unquantified heterogeneity of the environmental parameters across microarray experiments frustrates regulatory network inference. We propose an inference algorithm that models the influence of environmental parameters on gene expression. The approach is based on a yeast microarray compendium of chemostat steady-state experiments. Chemostat cultivation enables the accurate control and measurement of many of the key cultivation parameters, such as nutrient concentrations, growth rate and temperature. The observed transcript levels are explained by inferring the activity of TFs in response to combinations of cultivation parameters. The interplay between activated enhancers and repressors that bind a gene promoter determine the possible up- or downregulation of the gene. The model is translated into a linear integer optimization problem. The resulting regulatory network identifies the combinatorial effects of environmental parameters on TF activity and gene expression. The Matlab code is available from the authors upon request. Supplementary data are available at Bioinformatics online.

  7. Combinatorial influence of environmental parameters on transcription factor activity

    PubMed Central

    Knijnenburg, T.A.; Wessels, L.F.A.; Reinders, M.J.T.

    2008-01-01

    Motivation: Cells receive a wide variety of environmental signals, which are often processed combinatorially to generate specific genetic responses. Changes in transcript levels, as observed across different environmental conditions, can, to a large extent, be attributed to changes in the activity of transcription factors (TFs). However, in unraveling these transcription regulation networks, the actual environmental signals are often not incorporated into the model, simply because they have not been measured. The unquantified heterogeneity of the environmental parameters across microarray experiments frustrates regulatory network inference. Results: We propose an inference algorithm that models the influence of environmental parameters on gene expression. The approach is based on a yeast microarray compendium of chemostat steady-state experiments. Chemostat cultivation enables the accurate control and measurement of many of the key cultivation parameters, such as nutrient concentrations, growth rate and temperature. The observed transcript levels are explained by inferring the activity of TFs in response to combinations of cultivation parameters. The interplay between activated enhancers and repressors that bind a gene promoter determine the possible up- or downregulation of the gene. The model is translated into a linear integer optimization problem. The resulting regulatory network identifies the combinatorial effects of environmental parameters on TF activity and gene expression. Availability: The Matlab code is available from the authors upon request. Contact: t.a.knijnenburg@tudelft.nl Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18586711

  8. Transcriptional Regulation in Saccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

    PubMed Central

    Hahn, Steven; Young, Elton T.

    2011-01-01

    Here we review recent advances in understanding the regulation of mRNA synthesis in Saccharomyces cerevisiae. Many fundamental gene regulatory mechanisms have been conserved in all eukaryotes, and budding yeast has been at the forefront in the discovery and dissection of these conserved mechanisms. Topics covered include upstream activation sequence and promoter structure, transcription factor classification, and examples of regulated transcription factor activity. We also examine advances in understanding the RNA polymerase II transcription machinery, conserved coactivator complexes, transcription activation domains, and the cooperation of these factors in gene regulatory mechanisms. PMID:22084422

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

    PubMed

    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.

  10. Methylation of transcription factor YY2 regulates its transcriptional activity and cell proliferation

    PubMed Central

    Wu, Xiao-nan; Shi, Tao-tao; He, Yao-hui; Wang, Fei-fei; Sang, Rui; Ding, Jian-cheng; Zhang, Wen-juan; Shu, Xing-yi; Shen, Hai-feng; Yi, Jia; Gao, Xiang; Liu, Wen

    2017-01-01

    Yin Yang 1 (YY1) is a multifunctional DNA-binding transcription factor shown to be critical in a variety of biological processes, and its activity and function have been shown to be regulated by multitude of mechanisms, which include but are not limited to post-translational modifications (PTMs), its associated proteins and cellular localization. YY2, the paralog of YY1 in mouse and human, has been proposed to function redundantly or oppositely in a context-specific manner compared with YY1. Despite its functional importance, how YY2’s DNA-binding activity and function are regulated, particularly by PTMs, remains completely unknown. Here we report the first PTM with functional characterization on YY2, namely lysine 247 monomethylation (K247me1), which was found to be dynamically regulated by SET7/9 and LSD1 both in vitro and in cultured cells. Functional study revealed that SET7/9-mediated YY2 methylation regulated its DNA-binding activity in vitro and in association with chromatin examined by chromatin immunoprecipitation coupled with sequencing (ChIP-seq) in cultured cells. Knockout of YY2, SET7/9 or LSD1 by CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9-mediated gene editing followed by RNA sequencing (RNA-seq) revealed that a subset of genes was positively regulated by YY2 and SET7/9, but negatively regulated by LSD1, which were enriched with genes involved in cell proliferation regulation. Importantly, YY2-regulated gene transcription, cell proliferation and tumor growth were dependent, at least partially, on YY2 K247 methylation. Finally, somatic mutations on YY2 found in cancer, which are in close proximity to K247, altered its methylation, DNA-binding activity and gene transcription it controls. Our findings revealed the first PTM with functional implications imposed on YY2 protein, and linked YY2 methylation with its biological functions. PMID:29098080

  11. E2F transcription factors and digestive system malignancies: how much do we know?

    PubMed

    Xanthoulis, Athanasios; Tiniakos, Dina G

    2013-06-07

    E2F family of transcription factors regulates various cellular functions related to cell cycle and apoptosis. Its individual members have traditionally been classified into activators and repressors, based on in vitro studies. However their contribution in human cancer is more complicated and difficult to predict. We review current knowledge on the expression of E2Fs in digestive system malignancies and its clinical implications for patient prognosis and treatment. E2F1, the most extensively studied member and the only one with prognostic value, exhibits a tumor-suppressing activity in esophageal, gastric and colorectal adenocarcinoma, and in hepatocellular carcinoma (HCC), whereas in pancreatic ductal adenocarcinoma and esophageal squamous cell carcinoma may function as a tumor-promoter. In the latter malignancies, E2F1 immunohistochemical expression has been correlated with higher tumor grade and worse patient survival, whereas in esophageal, gastric and colorectal adenocarcinomas is a marker of increased patient survival. E2F2 has only been studied in colorectal cancer, where its role is not considered significant. E2F4's role in colorectal, gastric and hepatic carcinogenesis is tumor-promoting. E2F8 is strongly upregulated in human HCC, thus possibly contributing to hepatocarcinogenesis. Adenoviral transfer of E2F as gene therapy to sensitize pancreatic cancer cells for chemotherapeutic agents has been used in experimental studies. Other therapeutic strategies are yet to be developed, but it appears that targeted approaches using E2F-agonists or antagonists should take into account the tissue-dependent function of each E2F member. Further understanding of E2Fs' contribution in cellular functions in vivo would help clarify their role in carcinogenesis.

  12. Repression of TFIIH Transcriptional Activity and TFIIH-Associated cdk7 Kinase Activity at Mitosis

    PubMed Central

    Long, John J.; Leresche, Anne; Kriwacki, Richard W.; Gottesfeld, Joel M.

    1998-01-01

    Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis. PMID:9488463

  13. Distinct Contributions of Conserved Modules to Runt Transcription Factor Activity

    PubMed Central

    Walrad, Pegine B.; Hang, Saiyu; Joseph, Genevieve S.; Salas, Julia

    2010-01-01

    Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators. PMID:20462957

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

    PubMed

    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. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Global RNA association with the transcriptionally active chromosome of chloroplasts.

    PubMed

    Lehniger, Marie-Kristin; Finster, Sabrina; Melonek, Joanna; Oetke, Svenja; Krupinska, Karin; Schmitz-Linneweber, Christian

    2017-10-01

    Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

  16. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast.

    PubMed

    Skjoedt, Mette L; Snoek, Tim; Kildegaard, Kanchana R; Arsovska, Dushica; Eichenberger, Michael; Goedecke, Tobias J; Rajkumar, Arun S; Zhang, Jie; Kristensen, Mette; Lehka, Beata J; Siedler, Solvej; Borodina, Irina; Jensen, Michael K; Keasling, Jay D

    2016-11-01

    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,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications.

  17. Zipper plot: visualizing transcriptional activity of genomic regions.

    PubMed

    Avila Cobos, Francisco; Anckaert, Jasper; Volders, Pieter-Jan; Everaert, Celine; Rombaut, Dries; Vandesompele, Jo; De Preter, Katleen; Mestdagh, Pieter

    2017-05-02

    Reconstructing transcript models from RNA-sequencing (RNA-seq) data and establishing these as independent transcriptional units can be a challenging task. Current state-of-the-art tools for long non-coding RNA (lncRNA) annotation are mainly based on evolutionary constraints, which may result in false negatives due to the overall limited conservation of lncRNAs. To tackle this problem we have developed the Zipper plot, a novel visualization and analysis method that enables users to simultaneously interrogate thousands of human putative transcription start sites (TSSs) in relation to various features that are indicative for transcriptional activity. These include publicly available CAGE-sequencing, ChIP-sequencing and DNase-sequencing datasets. Our method only requires three tab-separated fields (chromosome, genomic coordinate of the TSS and strand) as input and generates a report that includes a detailed summary table, a Zipper plot and several statistics derived from this plot. Using the Zipper plot, we found evidence of transcription for a set of well-characterized lncRNAs and observed that fewer mono-exonic lncRNAs have CAGE peaks overlapping with their TSSs compared to multi-exonic lncRNAs. Using publicly available RNA-seq data, we found more than one hundred cases where junction reads connected protein-coding gene exons with a downstream mono-exonic lncRNA, revealing the need for a careful evaluation of lncRNA 5'-boundaries. Our method is implemented using the statistical programming language R and is freely available as a webtool.

  18. Global transcriptional regulatory network for Escherichia coli robustly connects gene expression to transcription factor activities

    PubMed Central

    Fang, Xin; Sastry, Anand; Mih, Nathan; Kim, Donghyuk; Tan, Justin; Lloyd, Colton J.; Gao, Ye; Yang, Laurence; Palsson, Bernhard O.

    2017-01-01

    Transcriptional regulatory networks (TRNs) have been studied intensely for >25 y. Yet, even for the Escherichia coli TRN—probably the best characterized TRN—several questions remain. Here, we address three questions: (i) How complete is our knowledge of the E. coli TRN; (ii) how well can we predict gene expression using this TRN; and (iii) how robust is our understanding of the TRN? First, we reconstructed a high-confidence TRN (hiTRN) consisting of 147 transcription factors (TFs) regulating 1,538 transcription units (TUs) encoding 1,764 genes. The 3,797 high-confidence regulatory interactions were collected from published, validated chromatin immunoprecipitation (ChIP) data and RegulonDB. For 21 different TF knockouts, up to 63% of the differentially expressed genes in the hiTRN were traced to the knocked-out TF through regulatory cascades. Second, we trained supervised machine learning algorithms to predict the expression of 1,364 TUs given TF activities using 441 samples. The algorithms accurately predicted condition-specific expression for 86% (1,174 of 1,364) of the TUs, while 193 TUs (14%) were predicted better than random TRNs. Third, we identified 10 regulatory modules whose definitions were robust against changes to the TRN or expression compendium. Using surrogate variable analysis, we also identified three unmodeled factors that systematically influenced gene expression. Our computational workflow comprehensively characterizes the predictive capabilities and systems-level functions of an organism’s TRN from disparate data types. PMID:28874552

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

    PubMed Central

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

    2009-01-01

    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. PMID:19644446

  20. Transcriptionally Active Heterochromatin in Rye B Chromosomes[W

    PubMed Central

    Carchilan, Mariana; Delgado, Margarida; Ribeiro, Teresa; Costa-Nunes, Pedro; Caperta, Ana; Morais-Cecílio, Leonor; Jones, R. Neil; Viegas, Wanda; Houben, Andreas

    2007-01-01

    B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type–dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA. PMID:17586652

  1. Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae

    PubMed Central

    Erkes, Annett; Reschke, Maik; Boch, Jens

    2017-01-01

    Abstract Transcription activator-like effectors (TALEs) are secreted by plant–pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity. PMID:28637323

  2. Exosomes from uninfected cells activate transcription of latent HIV-1.

    PubMed

    Barclay, Robert A; Schwab, Angela; DeMarino, Catherine; Akpamagbo, Yao; Lepene, Benjamin; Kassaye, Seble; Iordanskiy, Sergey; Kashanchi, Fatah

    2017-07-14

    HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1-infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR- gag , can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo , especially in the presence of combination antiretroviral therapy.

  3. The co-existence of transcriptional activator and transcriptional repressor MEF2 complexes influences tumor aggressiveness

    PubMed Central

    Di Giorgio, Eros; Franforte, Elisa; Cefalù, Sebastiano; Rossi, Sabrina; Dei Tos, Angelo Paolo; Polano, Maurizio; Maestro, Roberta; Paluvai, Harikrishnareddy

    2017-01-01

    The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS. PMID:28419090

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

    PubMed Central

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

    2016-01-01

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

  5. COACTIVATOR ACTIVATOR (CoAA) PREVENTS THE TRANSCRIPTIONAL ACTIVITY OF RUNT DOMAIN TRANSCRIPTION FACTORS

    PubMed Central

    Li, Xiaodong; Hoeppner, Luke H.; Jensen, Eric D.; Gopalakrishnan, Rajaram; Westendorf, Jennifer J.

    2013-01-01

    Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells. PMID:19585539

  6. Activation of p53 Transcriptional Activity by SMRT: a Histone Deacetylase 3-Independent Function of a Transcriptional Corepressor

    PubMed Central

    Adikesavan, Anbu Karani; Karmakar, Sudipan; Pardo, Patricia; Wang, Liguo; Liu, Shuang; Li, Wei

    2014-01-01

    The silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) is an established histone deacetylase 3 (HDAC3)-dependent transcriptional corepressor. Microarray analyses of MCF-7 cells transfected with control or SMRT small interfering RNA revealed SMRT regulation of genes involved in DNA damage responses, and the levels of the DNA damage marker γH2AX as well as poly(ADP-ribose) polymerase cleavage were elevated in SMRT-depleted cells treated with doxorubicin. A number of these genes are established p53 targets. SMRT knockdown decreased the activity of two p53-dependent reporter genes as well as the expression of p53 target genes, such as CDKN1A (which encodes p21). SMRT bound directly to p53 and was recruited to p53 binding sites within the p21 promoter. Depletion of GPS2 and TBL1, components of the SMRT corepressor complex, but not histone deacetylase 3 (HDAC3) decreased p21-luciferase activity. p53 bound to the SMRT deacetylase activation domain (DAD), which mediates HDAC3 binding and activation, and HDAC3 could attenuate p53 binding to the DAD region of SMRT. Moreover, an HDAC3 binding-deficient SMRT DAD mutant coactivated p53 transcriptional activity. Collectively, these data highlight a biological role for SMRT in mediating DNA damage responses and suggest a model where p53 binding to the DAD limits HDAC3 interaction with this coregulator, thereby facilitating SMRT coactivation of p53-dependent gene expression. PMID:24449765

  7. Tracking transcriptional activities with high-content epifluorescent imaging

    NASA Astrophysics Data System (ADS)

    Hua, Jianping; Sima, Chao; Cypert, Milana; Gooden, Gerald C.; Shack, Sonsoles; Alla, Lalitamba; Smith, Edward A.; Trent, Jeffrey M.; Dougherty, Edward R.; Bittner, Michael L.

    2012-04-01

    High-content cell imaging based on fluorescent protein reporters has recently been used to track the transcriptional activities of multiple genes under different external stimuli for extended periods. This technology enhances our ability to discover treatment-induced regulatory mechanisms, temporally order their onsets and recognize their relationships. To fully realize these possibilities and explore their potential in biological and pharmaceutical applications, we introduce a new data processing procedure to extract information about the dynamics of cell processes based on this technology. The proposed procedure contains two parts: (1) image processing, where the fluorescent images are processed to identify individual cells and allow their transcriptional activity levels to be quantified; and (2) data representation, where the extracted time course data are summarized and represented in a way that facilitates efficient evaluation. Experiments show that the proposed procedure achieves fast and robust image segmentation with sufficient accuracy. The extracted cellular dynamics are highly reproducible and sensitive enough to detect subtle activity differences and identify mechanisms responding to selected perturbations. This method should be able to help biologists identify the alterations of cellular mechanisms that allow drug candidates to change cell behavior and thereby improve the efficiency of drug discovery and treatment design.

  8. Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

    PubMed Central

    Cerqueira, Gustavo M.; Souza, Natalie M.; Araújo, Eduardo R.; Barros, Aline T.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Nascimento, Ana L. T. O.

    2011-01-01

    Background Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. Methodology and Principal Findings A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. Conclusions The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa. PMID:21445252

  9. Sumoylation activates the transcriptional activity of Pax-6, an important transcription factor for eye and brain development

    PubMed Central

    Yan, Qin; Gong, Lili; Deng, Mi; Zhang, Lan; Sun, Shuming; Liu, Jiao; Ma, Haili; Yuan, Dan; Chen, Pei-Chao; Hu, Xiaohui; Liu, Jinping; Qin, Jichao; Xiao, Ling; Huang, Xiao-Qin; Zhang, Jian; Wan-Cheng Li, David

    2010-01-01

    Pax-6 is an evolutionarily conserved transcription factor regulating brain and eye development. Four Pax-6 isoforms have been reported previously. Although the longer Pax-6 isoforms (p46 and p48) bear two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), the shorter Pax-6 isoform p32 contains only the HD for DNA binding. Although a third domain, the proline-, serine- and threonine-enriched activation (PST) domain, in the C termini of all Pax-6 isoforms mediates their transcriptional modulation via phosphorylation, how p32 Pax-6 could regulate target genes remains to be elucidated. In the present study, we show that sumoylation at K91 is required for p32 Pax-6 to bind to a HD-specific site and regulate expression of target genes. First, in vitro-synthesized p32 Pax-6 alone cannot bind the P3 sequence, which contains the HD recognition site, unless it is preincubated with nuclear extracts precleared by anti–Pax-6 but not by anti-small ubiquitin-related modifier 1 (anti-SUMO1) antibody. Second, in vitro-synthesized p32 Pax-6 can be sumoylated by SUMO1, and the sumoylated p32 Pax-6 then can bind to the P3 sequence. Third, Pax-6 and SUMO1 are colocalized in the embryonic optic and lens vesicles and can be coimmunoprecipitated. Finally, SUMO1-conjugated p32 Pax-6 exists in both the nucleus and cytoplasm, and sumoylation significantly enhances the DNA-binding ability of p32 Pax-6 and positively regulates gene expression. Together, our results demonstrate that sumoylation activates p32 Pax-6 in both DNA-binding and transcriptional activities. In addition, our studies demonstrate that p32 and p46 Pax-6 possess differential DNA-binding and regulatory activities. PMID:21084637

  10. The Transcription Factor p53 Influences Microglial Activation Phenotype

    PubMed Central

    Jayadev, Suman; Nesser, Nicole K.; Hopkins, Stephanie; Myers, Scott J.; Case, Amanda; Lee, Rona J.; Seaburg, Luke A.; Uo, Takuma; Murphy, Sean P.; Morrison, Richard S.; Garden, Gwenn A.

    2011-01-01

    Several neurodegenerative diseases are influenced by the innate immune response in the central nervous system (CNS). Microglia, have pro-inflammatory and subsequently neurotoxic actions as well as anti-inflammatory functions that promote recovery and repair. Very little is known about the transcriptional control of these specific microglial behaviors. We have previously shown that in HIV associated neurocognitive disorders (HAND), the transcription factor p53 accumulates in microglia and that microglial p53 expression is required for the in vitro neurotoxicity of the HIV coat glycoprotein gp120. These findings suggested a novel function for p53 in regulating microglial activation. Here we report that in the absence of p53, microglia demonstrate a blunted response to interferon-γ, failing to increase expression of genes associated with classical macrophage activation or secrete pro-inflammatory cytokines. Microarray analysis of global gene expression profiles revealed increased expression of genes associated with anti-inflammatory functions, phagocytosis and tissue repair in p53 knockout (p53−/−) microglia compared with those cultured from strain matched p53 expressing (p53+/+) mice. We further observed that p53−/− microglia demonstrate increased phagocytic activity in vitro and expression of markers for alternative macrophage activation both in vitro and in vivo. In HAND brain tissue, the alternative activation marker CD163 was expressed in a separate subset of microglia than those demonstrating p53 accumulation. These data suggest that p53 influences microglial behavior, supporting the adoption of a pro-inflammatory phenotype, while p53 deficiency promotes phagocytosis and gene expression associated with alternative activation and anti-inflammatory functions. PMID:21598312

  11. Mediator Undergoes a Compositional Change during Transcriptional Activation.

    PubMed

    Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

    2016-11-03

    Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Epigenetic priors for identifying active transcription factor binding sites.

    PubMed

    Cuellar-Partida, Gabriel; Buske, Fabian A; McLeay, Robert C; Whitington, Tom; Noble, William Stafford; Bailey, Timothy L

    2012-01-01

    Accurate knowledge of the genome-wide binding of transcription factors in a particular cell type or under a particular condition is necessary for understanding transcriptional regulation. Using epigenetic data such as histone modification and DNase I, accessibility data has been shown to improve motif-based in silico methods for predicting such binding, but this approach has not yet been fully explored. We describe a probabilistic method for combining one or more tracks of epigenetic data with a standard DNA sequence motif model to improve our ability to identify active transcription factor binding sites (TFBSs). We convert each data type into a position-specific probabilistic prior and combine these priors with a traditional probabilistic motif model to compute a log-posterior odds score. Our experiments, using histone modifications H3K4me1, H3K4me3, H3K9ac and H3K27ac, as well as DNase I sensitivity, show conclusively that the log-posterior odds score consistently outperforms a simple binary filter based on the same data. We also show that our approach performs competitively with a more complex method, CENTIPEDE, and suggest that the relative simplicity of the log-posterior odds scoring method makes it an appealing and very general method for identifying functional TFBSs on the basis of DNA and epigenetic evidence. FIMO, part of the MEME Suite software toolkit, now supports log-posterior odds scoring using position-specific priors for motif search. A web server and source code are available at http://meme.nbcr.net. Utilities for creating priors are at http://research.imb.uq.edu.au/t.bailey/SD/Cuellar2011. t.bailey@uq.edu.au Supplementary data are available at Bioinformatics online.

  13. Monitoring transcription initiation activities in rat and dog.

    PubMed

    Lizio, Marina; Mukarram, Abdul Kadir; Ohno, Mizuho; Watanabe, Shoko; Itoh, Masayoshi; Hasegawa, Akira; Lassmann, Timo; Severin, Jessica; Harshbarger, Jayson; Abugessaisa, Imad; Kasukawa, Takeya; Hon, Chung Chau; Carninci, Piero; Hayashizaki, Yoshihide; Forrest, Alistair R R; Kawaji, Hideya

    2017-11-28

    The promoter landscape of several non-human model organisms is far from complete. As a part of FANTOM5 data collection, we generated 13 profiles of transcription initiation activities in dog and rat aortic smooth muscle cells, mesenchymal stem cells and hepatocytes by employing CAGE (Cap Analysis of Gene Expression) technology combined with single molecule sequencing. Our analyses show that the CAGE profiles recapitulate known transcription start sites (TSSs) consistently, in addition to uncover novel TSSs. Our dataset can be thus used with high confidence to support gene annotation in dog and rat species. We identified 28,497 and 23,147 CAGE peaks, or promoter regions, for rat and dog respectively, and associated them to known genes. This approach could be seen as a standard method for improvement of existing gene models, as well as discovery of novel genes. Given that the FANTOM5 data collection includes dog and rat matched cell types in human and mouse as well, this data would also be useful for cross-species studies.

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

    SciTech Connect

    Shlomai, Amir, E-mail: amirsh@tasmc.health.gov.il; Institute for Gastroenterology and Liver disease, Tel-Aviv Sourasky Medical Center, 6 Weizmann street, Tel-Aviv; Shaul, Yosef

    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 enhancedmore » 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.« less

  15. Berberine Suppresses Adipocyte Differentiation via Decreasing CREB Transcriptional Activity

    PubMed Central

    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. PMID:25928058

  16. Comprehensive Behavioral Analysis of Activating Transcription Factor 5-Deficient Mice

    PubMed Central

    Umemura, Mariko; Ogura, Tae; Matsuzaki, Ayako; Nakano, Haruo; Takao, Keizo; Miyakawa, Tsuyoshi; Takahashi, Yuji

    2017-01-01

    Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5-/-) mice demonstrated abnormal olfactory bulb development due to impaired interneuron supply. Furthermore, ATF5-/- mice were less aggressive than ATF5+/+ mice. Although ATF5 is widely expressed in the brain, and involved in the regulation of proliferation and development of neurons, the physiological role of ATF5 in the higher brain remains unknown. Our objective was to investigate the physiological role of ATF5 in the higher brain. We performed a comprehensive behavioral analysis using ATF5-/- mice and wild type littermates. ATF5-/- mice exhibited abnormal locomotor activity in the open field test. They also exhibited abnormal anxiety-like behavior in the light/dark transition test and open field test. Furthermore, ATF5-/- mice displayed reduced social interaction in the Crawley’s social interaction test and increased pain sensitivity in the hot plate test compared with wild type. Finally, behavioral flexibility was reduced in the T-maze test in ATF5-/- mice compared with wild type. In addition, we demonstrated that ATF5-/- mice display disturbances of monoamine neurotransmitter levels in several brain regions. These results indicate that ATF5 deficiency elicits abnormal behaviors and the disturbance of monoamine neurotransmitter levels in the brain. The behavioral abnormalities of ATF5-/- mice may be due to the disturbance of monoamine levels. Taken together, these findings suggest that ATF5-/- mice may be a unique animal model of some psychiatric disorders. PMID:28744205

  17. ROBNCA: robust network component analysis for recovering transcription factor activities.

    PubMed

    Noor, Amina; Ahmad, Aitzaz; Serpedin, Erchin; Nounou, Mohamed; Nounou, Hazem

    2013-10-01

    Network component analysis (NCA) is an efficient method of reconstructing the transcription factor activity (TFA), which makes use of the gene expression data and prior information available about transcription factor (TF)-gene regulations. Most of the contemporary algorithms either exhibit the drawback of inconsistency and poor reliability, or suffer from prohibitive computational complexity. In addition, the existing algorithms do not possess the ability to counteract the presence of outliers in the microarray data. Hence, robust and computationally efficient algorithms are needed to enable practical applications. We propose ROBust Network Component Analysis (ROBNCA), a novel iterative algorithm that explicitly models the possible outliers in the microarray data. An attractive feature of the ROBNCA algorithm is the derivation of a closed form solution for estimating the connectivity matrix, which was not available in prior contributions. The ROBNCA algorithm is compared with FastNCA and the non-iterative NCA (NI-NCA). ROBNCA estimates the TF activity profiles as well as the TF-gene control strength matrix with a much higher degree of accuracy than FastNCA and NI-NCA, irrespective of varying noise, correlation and/or amount of outliers in case of synthetic data. The ROBNCA algorithm is also tested on Saccharomyces cerevisiae data and Escherichia coli data, and it is observed to outperform the existing algorithms. The run time of the ROBNCA algorithm is comparable with that of FastNCA, and is hundreds of times faster than NI-NCA. The ROBNCA software is available at http://people.tamu.edu/∼amina/ROBNCA

  18. Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains.

    PubMed Central

    Tell, G; Perrone, L; Fabbro, D; Pellizzari, L; Pucillo, C; De Felice, M; Acquaviva, R; Formisano, S; Damante, G

    1998-01-01

    The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development. PMID:9425125

  19. Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions

    PubMed Central

    Scholes, Natalie S.; Weinzierl, Robert O. J.

    2016-01-01

    Transcriptional activation domains (ADs) are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD) simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators. PMID:27175900

  20. Activating transcription factor 4 regulates stearate-induced vascular calcification.

    PubMed

    Masuda, Masashi; Ting, Tabitha C; Levi, Moshe; Saunders, Sommer J; Miyazaki-Anzai, Shinobu; Miyazaki, Makoto

    2012-08-01

    Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate.

  1. Activating transcription factor 4 regulates stearate-induced vascular calcification

    PubMed Central

    Masuda, Masashi; Ting, Tabitha C.; Levi, Moshe; Saunders, Sommer J.; Miyazaki-Anzai, Shinobu; Miyazaki, Makoto

    2012-01-01

    Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate. PMID:22628618

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

    EPA Science Inventory

    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...

  3. Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

    PubMed

    Sato, Takeo; Maekawa, Shugo; Konishi, Mineko; Yoshioka, Nozomi; Sasaki, Yuki; Maeda, Haruna; Ishida, Tetsuya; Kato, Yuki; Yamaguchi, Junji; Yanagisawa, Shuichi

    2017-01-29

    Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Transcriptional activation of Mina by Sp1/3 factors.

    PubMed

    Lian, Shangli; Potula, Hari Hara S K; Pillai, Meenu R; Van Stry, Melanie; Koyanagi, Madoka; Chung, Linda; Watanabe, Makiko; Bix, Mark

    2013-01-01

    Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

  5. Transcriptional Activation of Mina by Sp1/3 Factors

    PubMed Central

    Lian, Shangli; Potula, Hari Hara S. K.; Pillai, Meenu R.; Van Stry, Melanie; Koyanagi, Madoka; Chung, Linda; Watanabe, Makiko; Bix, Mark

    2013-01-01

    Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5′ region [1]. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays – reporter, gel shift and chromatin immunoprecipitation – to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways. PMID:24324617

  6. Nucleophosmin regulates the stability and transcriptional activity of p53.

    PubMed

    Colombo, Emanuela; Marine, Jean-Christophe; Danovi, Davide; Falini, Brunangelo; Pelicci, Pier Giuseppe

    2002-07-01

    Nucleophosmin (NPM) is a ubiquitously expressed nucleolar phosphoprotein that continuously shuttles between the nucleus and cytoplasm. It has been proposed to function in ribosomal protein assembly and transport, and also as a molecular chaperone that prevents proteins from aggregating in the crowded environment of the nucleolus. The NPM gene is involved in several tumour-associated chromosome translocations, which have resulted in the formation of fusion proteins that retain the amino terminus of NPM, including NPM ALK, NPM RAR and NPM MLF1 (ref. 6). It is generally thought that the NPM component is not involved in the transforming potential of these fusion proteins, but instead provides a dimerization interface for the oligomerization and the oncogenic conversion of the various NPM partners (ALK, RAR, MLF1). Here we show that NPM interacts directly with the tumour suppressor p53, regulates the increase in stability and transcriptional activation of p53 after different types of stress, and induces p53-dependent premature senescence on overexpression in diploid fibroblasts. These findings indicate that NPM is a crucial regulator of p53 and suggest that alterations of the NPM function by NPM fusion proteins might lead to deregulation of p53 in tumours.

  7. Activating Transcription Factor 3 Regulates Immune and Metabolic Homeostasis

    PubMed Central

    Rynes, Jan; Donohoe, Colin D.; Frommolt, Peter; Brodesser, Susanne; Jindra, Marek

    2012-01-01

    Integration of metabolic and immune responses during animal development ensures energy balance, permitting both growth and defense. Disturbed homeostasis causes organ failure, growth retardation, and metabolic disorders. Here, we show that the Drosophila melanogaster activating transcription factor 3 (Atf3) safeguards metabolic and immune system homeostasis. Loss of Atf3 results in chronic inflammation and starvation responses mounted primarily by the larval gut epithelium, while the fat body suffers lipid overload, causing energy imbalance and death. Hyperactive proinflammatory and stress signaling through NF-κB/Relish, Jun N-terminal kinase, and FOXO in atf3 mutants deregulates genes important for immune defense, digestion, and lipid metabolism. Reducing the dose of either FOXO or Relish normalizes both lipid metabolism and gene expression in atf3 mutants. The function of Atf3 is conserved, as human ATF3 averts some of the Drosophila mutant phenotypes, improving their survival. The single Drosophila Atf3 may incorporate the diversified roles of two related mammalian proteins. PMID:22851689

  8. TRIC: Capturing the direct cellular targets of promoter‐bound transcriptional activators

    PubMed Central

    Dugan, Amanda; Pricer, Rachel; Katz, Micah

    2016-01-01

    Abstract Transcriptional activators coordinate the dynamic assembly of multiprotein coactivator complexes required for gene expression to occur. Here we combine the power of in vivo covalent chemical capture with p‐benzoyl‐L‐phenylalanine (Bpa), a genetically incorporated photo‐crosslinking amino acid, and chromatin immunoprecipitation (ChIP) to capture the direct protein interactions of the transcriptional activator VP16 with the general transcription factor TBP at the GAL1 promoter in live yeast. PMID:27213278

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

    USDA-ARS?s Scientific Manuscript database

    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...

  10. Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells.

    PubMed Central

    Tower, J; Sollner-Webb, B

    1988-01-01

    Extracts of cells that are down-regulated for transcription by RNA polymerase I and RNA polymerase III exhibit a reduced in vitro transcriptional capacity. We have recently demonstrated that the down-regulation of polymerase I transcription in extracts of cycloheximide-treated and stationary-phase cells results from a lack of an activated subform of RNA polymerase I which is essential for rDNA transcription. To examine whether polymerase III transcriptional down-regulation occurs by a similar mechanism, the polymerase III transcription factors were isolated and added singly and in pairs to control cell extracts and to extracts of cells that had reduced polymerase III transcriptional activity due to cycloheximide treatment or growth into stationary phase. These down-regulations result from a specific reduction in TFIIIB; TFIIIC and polymerase III activities remain relatively constant. Thus, although transcription by both polymerase III and polymerase I is substantially decreased in extracts of growth-arrested cells, this regulation is brought about by reduction of different kinds of activities: a component of the polymerase III stable transcription complex in the former case and the activated subform of RNA polymerase I in the latter. Images PMID:3352599

  11. Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

    PubMed

    Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

    2013-10-17

    Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

  12. The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

    PubMed

    Hale, T K; Braithwaite, A W

    1999-08-20

    Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.

  13. Transcription co-activator SAYP mediates the action of STAT activator

    PubMed Central

    Panov, Vladislav V.; Kuzmina, Julia L.; Doronin, Semen A.; Kopantseva, Marina R.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Vorobyeva, Nadezhda E.; Shidlovskii, Yulii V.

    2012-01-01

    Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT. PMID:22123744

  14. Transcription co-activator SAYP mediates the action of STAT activator.

    PubMed

    Panov, Vladislav V; Kuzmina, Julia L; Doronin, Semen A; Kopantseva, Marina R; Nabirochkina, Elena N; Georgieva, Sofia G; Vorobyeva, Nadezhda E; Shidlovskii, Yulii V

    2012-03-01

    Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

  15. Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis.

    PubMed

    Li, S; Zhang, P; Zhang, M; Fu, C; Yu, L

    2013-01-01

    Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

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

    EPA Science Inventory

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

  17. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

    PubMed

    Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-02-07

    bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

  18. Pokemon decreases the transcriptional activity of RARα in the absence of ligand.

    PubMed

    Yang, Yutao; Li, Yueting; Di, Fei; Cui, Jiajun; Wang, Yue; David Xu, Zhi-Qing

    2016-12-20

    Pokemon is a transcriptional repressor that belongs to the POZ and Krüppel (POK) protein family. In this study, we investigated the potential interaction between Pokemon and retinoic acid receptor alpha (RARα) and determined the role of Pokemon in regulation of RARα transcriptional activity in the absence of ligand. We found that Pokemon could directly interact with RARα. Moreover, we demonstrated that Pokemon could decrease the transcriptional activity of RARα in the absence of ligand. Furthermore, we showed that Pokemon could repress the transcriptional activity of RARα by increasing the recruitment of nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the retinoic acid response element (RARE) element. Taken together, these data suggest that Pokemon is a novel partner of RARα that acts as a co-repressor to regulate RARα transcriptional activity in the absence of ligand.

  19. SKN-1-independent transcriptional activation of glutathione S-transferase 4 (GST-4) by EGF signaling

    PubMed Central

    Van de Walle, Pieter; Schoofs, Liliane

    2016-01-01

    ABSTRACT In C. elegans research, transcriptional activation of glutathione S-transferase 4 (gst-4) is often used as a read-out for SKN-1 activity. While many heed an assumed non-exclusivity of the GFP reporter signal driven by the gst-4 promoter to SKN-1, this is also often ignored. We here show that gst-4 can also be transcriptionally activated by EOR-1, a transcription factor mediating effects of the epidermal growth factor (EGF) pathway. Along with enhancing exogenous oxidative stress tolerance, EOR-1 inde-pendently of SKN-1 increases gst-4 transcription in response to augmented EGF signaling. Our findings caution researchers within the C. elegans community to always rely on sufficient experimental controls when assaying SKN-1 transcriptional activity with a gst-4p::gfp reporter, such as SKN-1 loss-of-function mutants and/or additional target genes next to gst-4. PMID:28090393

  20. Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

    PubMed Central

    Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I

    1991-01-01

    Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560

  1. SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity.

    PubMed

    Nishida, Tamotsu; Yamada, Yoshiji

    2016-05-13

    Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Mitochondrial genome-maintaining activity of mouse mitochondrial transcription factor A and its transcript isoform in Saccharomyces cerevisiae.

    PubMed

    Yoon, Young Geol; Koob, Michael D; Yoo, Young Hyun

    2011-09-15

    Mitochondrial transcription factor A (Tfam) binds to and organizes mitochondrial DNA (mtDNA) genome into a mitochondrial nucleoid (mt-nucleoid) structure, which is necessary for mtDNA transcription and maintenance. Here, we demonstrate the mtDNA-organizing activity of mouse Tfam and its transcript isoform (Tfam(iso)), which has a smaller high-mobility group (HMG)-box1 domain, using a yeast model system that contains a deletion of the yeast homolog of mouse Tfam protein, Abf2p. When the mouse Tfam genes were introduced into the ABF2 locus of yeast genome, the corresponding mouse proteins, Tfam and Tfam(iso), can functionally replace the yeast Abf2p and support mtDNA maintenance and mitochondrial biogenesis in yeast. Growth properties, mtDNA content and mitochondrial protein levels of genes encoded in the mtDNA were comparable in the strains expressing mouse proteins and the wild-type yeast strain, indicating that the proteins have robust mtDNA-maintaining and -expressing function in yeast mitochondria. These results imply that the mtDNA-organizing activities of the mouse mt-nucleoid proteins are structurally and evolutionary conserved, thus they can maintain the mtDNA of distantly related and distinctively different species, such as yeast. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Rapid activity-induced transcription of arc and other IEGs relies on poised RNA polymerase II

    PubMed Central

    Saha, Ramendra N.; Wissink, Erin M.; Bailey, Emma R.; Zhao, Meilan; Fargo, David C.; Hwang, Ji-yeon; Daigle, Kelly R.; Fenn, J. Daniel; Adelman, Karen; Dudek, Serena M.

    2011-01-01

    Summary Transcription of immediate early genes (IEGs) in neurons is exquisitely sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We demonstrate that several IEGs such as arc/arg3.1 are poised for near-instantaneous transcription by the stalling of RNA Polymerase II (Pol II) just downstream of the transcription start site in rat neurons. RNAi-depletion of Negative Elongation Factor, a mediator of Pol II stalling, reduces the Pol II occupancy of the arc promoter and compromises the rapid induction of arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that are expressed later and largely lack promoter proximal Pol II stalling. Together, our data strongly indicate that rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory. PMID:21623364

  4. Transcriptional regulation of cellular ageing by the CCAAT box-binding factor CBF/NF-Y.

    PubMed

    Matuoka, Koozi; Chen, Kuang Yu

    2002-09-01

    Cellular ageing is a systematic process affecting the entirety of cell structure and function. Since changes in gene expression are extensive and global during ageing, involvement of general transcription regulators in the phenomenon is likely. Here, we focus on NF-Y, the major CCAAT box-binding factor, which exerts differential regulation on a wide variety of genes through its interaction with the CCAAT box present in as many as 25% of the eukaryotic genes. When a cell ages, senescing signals arise, typically through DNA damage due to oxidative stress or telomere shortening, and are transduced to proteins such as p53, retinoblastoma protein, and phosphatidylinositol 3-kinase. Among them, activated p53 family proteins suppress the function of NF-Y and thereby downregulate a set of cell cycle-related genes, including E2F1, which further leads to downregulation of E2F-regulated genes and cell cycle arrest. The p53 family also induces other ageing phenotypes such as morphological alterations and senescence-associated beta-galactosidase (SA-gal) presumably by upregulation of some genes through NF-Y suppression. In fact, the activities of NF-Y and E2F decrease during ageing and a dominant negative NF-YA induces SA-gal. Based on these observations, NF-Y appears to play an important role in the process of cellular ageing.

  5. The yeast Hot1 transcription factor is critical for activating a single target gene, STL1

    PubMed Central

    Bai, Chen; Tesker, Masha; Engelberg, David

    2015-01-01

    Transcription factors are commonly activated by signal transduction cascades and induce expression of many genes. They therefore play critical roles in determining the cell's fate. The yeast Hog1 MAP kinase pathway is believed to control the transcription of hundreds of genes via several transcription factors. To identify the bona fide target genes of Hog1, we inducibly expressed the spontaneously active variant Hog1D170A+F318L in cells lacking the Hog1 activator Pbs2. This system allowed monitoring the effects of Hog1 by itself. Expression of Hog1D170A+F318L in pbs2∆ cells imposed induction of just 105 and suppression of only 26 transcripts by at least twofold. We looked for the Hog1-responsive element within the promoter of the most highly induced gene, STL1 (88-fold). A novel Hog1 responsive element (HoRE) was identified and shown to be the direct target of the transcription factor Hot1. Unexpectedly, we could not find this HoRE in any other yeast promoter. In addition, the only gene whose expression was abolished in hot1∆ cells was STL1. Thus Hot1 is essential for transcription of just one gene, STL1. Hot1 may represent a class of transcription factors that are essential for transcription of a very few genes or even just one. PMID:25904326

  6. RHON1 mediates a Rho-like activity for transcription termination in plastids of Arabidopsis thaliana.

    PubMed

    Chi, Wei; He, Baoye; Manavski, Nikolay; Mao, Juan; Ji, Daili; Lu, Congming; Rochaix, Jean David; Meurer, Jörg; Zhang, Lixin

    2014-12-01

    Although transcription termination is essential to generate functional RNAs, its underlying molecular mechanisms are still poorly understood in plastids of vascular plants. Here, we show that the RNA binding protein RHON1 participates in transcriptional termination of rbcL (encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) in Arabidopsis thaliana. Inactivation of RHON1 leads to enhanced rbcL read-through transcription and to aberrant accD (encoding β-subunit of the acetyl-CoA carboxylase) transcriptional initiation, which may result from inefficient transcription termination of rbcL. RHON1 can bind to the mRNA as well as to single-stranded DNA of rbcL, displays an RNA-dependent ATPase activity, and terminates transcription of rbcL in vitro. These results suggest that RHON1 terminates rbcL transcription using an ATP-driven mechanism similar to that of Rho of Escherichia coli. This RHON1-dependent transcription termination occurs in Arabidopsis but not in rice (Oryza sativa) and appears to reflect a fundamental difference between plastomes of dicotyledonous and monocotyledonous plants. Our results point to the importance and significance of plastid transcription termination and provide insights into its machinery in an evolutionary context. © 2014 American Society of Plant Biologists. All rights reserved.

  7. Role of the σ 54 Activator Interacting Domain in Bacterial Transcription Initiation

    SciTech Connect

    Siegel, Alexander R.; Wemmer, David E.

    Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ 54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA + ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ 54 are primarily made through anmore » N-terminal σ 54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ 54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ 54 alone. In this paper, we identified a minimal construct of the Aquifex aeolicus σ 54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1–σ 54 AID complex using NMR spectroscopy. We show that the σ 54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Finally, understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ 54 bacterial transcription initiation.« less

  8. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

    PubMed

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-06-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

  9. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

    PubMed Central

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-01-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

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

    EPA Science Inventory

    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. Simulation-Based Validation of the p53 Transcriptional Activity with Hybrid Functional Petri Net.

    PubMed

    Doi, Atsushi; Nagasaki, Masao; Matsuno, Hiroshi; Miyano, Satoru

    2011-01-01

    MDM2 and p19ARF are essential proteins in cancer pathways forming a complex with protein p53 to control the transcriptional activity of protein p53. It is confirmed that protein p53 loses its transcriptional activity by forming the functional dimer with protein MDM2. However, it is still unclear that protein p53 keeps its transcriptional activity when it forms the trimer with proteins MDM2 and p19ARF. We have observed mutual behaviors among genes p53, MDM2, p19ARF and their products on a computational model with hybrid functional Petri net (HFPN) which is constructed based on information described in the literature. The simulation results suggested that protein p53 should have the transcriptional activity in the forms of the trimer of proteins p53, MDM2, and p19ARF. This paper also discusses the advantages of HFPN based modeling method in terms of pathway description for simulations.

  12. Regulatory role of glycogen synthase kinase 3 for transcriptional activity of ADD1/SREBP1c.

    PubMed

    Kim, Kang Ho; Song, Min Jeong; Yoo, Eung Jae; Choe, Sung Sik; Park, Sang Dai; Kim, Jae Bum

    2004-12-10

    Adipocyte determination- and differentiation-dependent factor 1 (ADD1) plays important roles in lipid metabolism and insulin-dependent gene expression. Because insulin stimulates carbohydrate and lipid synthesis, it would be important to decipher how the transcriptional activity of ADD1/SREBP1c is regulated in the insulin signaling pathway. In this study, we demonstrated that glycogen synthase kinase (GSK)-3 negatively regulates the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitors enhanced a transcriptional activity of ADD1/SREBP1c and expression of ADD1/SREBP1c target genes including fatty acid synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and steroyl-CoA desaturase 1 (SCD1) in adipocytes and hepatocytes. In contrast, overexpression of GSK3beta down-regulated the transcriptional activity of ADD1/SREBP1c. GSK3 inhibitor-mediated ADD1/SREBP1c target gene activation did not require de novo protein synthesis, implying that GSK3 might affect transcriptional activity of ADD1/SREBP1c at the level of post-translational modification. Additionally, we demonstrated that GSK3 efficiently phosphorylated ADD1/SREBP1c in vitro and in vivo. Therefore, these data suggest that GSK3 inactivation is crucial to confer stimulated transcriptional activity of ADD1/SREBP1c for insulin-dependent gene expression, which would coordinate lipid and glucose metabolism.

  13. Global Identification and Characterization of Transcriptionally Active Regions in the Rice Genome

    PubMed Central

    Stolc, Viktor; Deng, Wei; He, Hang; Korbel, Jan; Chen, Xuewei; Tongprasit, Waraporn; Ronald, Pamela; Chen, Runsheng; Gerstein, Mark; Wang Deng, Xing

    2007-01-01

    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. PMID:17372628

  14. Transcriptionally active LTR retrotransposons in Eucalyptus genus are differentially expressed and insertionally polymorphic.

    PubMed

    Marcon, Helena Sanches; Domingues, Douglas Silva; Silva, Juliana Costa; Borges, Rafael Junqueira; Matioli, Fábio Filippi; Fontes, Marcos Roberto de Mattos; Marino, Celso Luis

    2015-08-14

    In Eucalyptus genus, studies on genome composition and transposable elements (TEs) are particularly scarce. Nearly half of the recently released Eucalyptus grandis genome is composed by retrotransposons and this data provides an important opportunity to understand TE dynamics in Eucalyptus genome and transcriptome. We characterized nine families of transcriptionally active LTR retrotransposons from Copia and Gypsy superfamilies in Eucalyptus grandis genome and we depicted genomic distribution and copy number in two Eucalyptus species. We also evaluated genomic polymorphism and transcriptional profile in three organs of five Eucalyptus species. We observed contrasting genomic and transcriptional behavior in the same family among different species. RLC_egMax_1 was the most prevalent family and RLC_egAngela_1 was the family with the lowest copy number. Most families of both superfamilies have their insertions occurring <3 million years, except one Copia family, RLC_egBianca_1. Protein theoretical models suggest different properties between Copia and Gypsy domains. IRAP and REMAP markers suggested genomic polymorphisms among Eucalyptus species. Using EST analysis and qRT-PCRs, we observed transcriptional activity in several tissues and in all evaluated species. In some families, osmotic stress increases transcript values. Our strategy was successful in isolating transcriptionally active retrotransposons in Eucalyptus, and each family has a particular genomic and transcriptional pattern. Overall, our results show that retrotransposon activity have differentially affected genome and transcriptome among Eucalyptus species.

  15. Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

    PubMed

    Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

    2005-04-15

    Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

  16. Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

    PubMed

    Deng, Jian-Jun; Kong, Ka-Yiu Edwin; Gao, Wei-Wei; Tang, Hei-Man Vincent; Chaudhary, Vidyanath; Cheng, Yun; Zhou, Jie; Chan, Chi-Ping; Wong, Danny Ka-Ho; Yuen, Man-Fung; Jin, Dong-Yan

    2017-04-01

    Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD + -dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. The PPARα/p16INK4a Pathway inhibits Vascular Smooth Muscle Cell Proliferation by repressing Cell Cycle-dependent Telomerase Activation

    PubMed Central

    Gizard, Florence; Nomiyama, Takashi; Zhao, Yue; Findeisen, Hannes M.; Heywood, Elizabeth B.; Jones, Karrie L.; Staels, Bart; Bruemmer, Dennis

    2009-01-01

    Peroxisome Proliferator-Activated Receptor (PPAR) α, the molecular target for fibrates used to treat dyslipidemia, exerts pleiotropic effects on vascular cells. In vascular smooth muscle cells (VSMCs), we have previously demonstrated that PPARα activation suppresses G1→S cell cycle progression by targeting the cyclin-dependent kinase inhibitor p16INK4a (p16). In the present study, we demonstrate that this inhibition of VSMC proliferation by PPARα is mediated through a p16-dependent suppression of telomerase activity, which has been implicated in key cellular functions including proliferation. PPARα activation inhibited mitogen-induced telomerase activity by repressing the catalytic subunit telomerase reverse transcriptase (TERT) through negative cross-talk with an E2F-1-dependent trans-activation of the TERT promoter. This trans-repression involved the recruitment of the retinoblastoma (RB) family proteins p107 and p130 to the TERT promoter resulting in impaired E2F-1 binding, an effect which was dependent on p16. The inhibition of cell proliferation by PPARα activation was lost in VSMC following TERT overexpression or knock-down, pointing to a key role of telomerase as a target for the antiproliferative effects of PPARα. Finally, we demonstrate that PPARα agonists suppress telomerase activation during the proliferative response following vascular injury indicating that these findings are applicable in vivo. In concert, these results demonstrate that the anti-proliferative effects of PPARα in VSMCs depend on the suppression of telomerase activity by targeting the p16/RB/E2F transcriptional cascade. PMID:18818403

  18. PARP-1 Controls the Adipogenic Transcriptional Program by PARylating C/EBPβ and Modulating Its Transcriptional Activity.

    PubMed

    Luo, Xin; Ryu, Keun Woo; Kim, Dae-Seok; Nandu, Tulip; Medina, Carlos J; Gupte, Rebecca; Gibson, Bryan A; Soccio, Raymond E; Yu, Yonghao; Gupta, Rana K; Kraus, W Lee

    2017-01-19

    Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by PARP family members, such as PARP-1. Although PARylation has been studied extensively, few examples of definitive biological roles for site-specific PARylation have been reported. Here we show that C/EBPβ, a key pro-adipogenic transcription factor, is PARylated by PARP-1 on three amino acids in a conserved regulatory domain. PARylation at these sites inhibits C/EBPβ's DNA binding and transcriptional activities and attenuates adipogenesis in various genetic and cell-based models. Interestingly, PARP-1 catalytic activity drops precipitously during the first 48 hr of differentiation, corresponding to a release of C/EBPβ from PARylation-mediated inhibition. This promotes the binding of C/EBPβ at enhancers controlling the expression of adipogenic target genes and continued differentiation. Depletion or chemical inhibition of PARP-1, or mutation of the PARylation sites on C/EBPβ, enhances these early adipogenic events. Collectively, our results provide a clear example of how site-specific PARylation drives biological outcomes. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator.

    PubMed

    Lee, Sooncheol; Nguyen, Huong Minh; Kang, Changwon

    2010-10-01

    No biological function has been identified for tiny RNA transcripts that are abortively and repetitiously released from initiation complexes of RNA polymerase in vitro and in vivo to date. In this study, we show that abortive initiation affects termination in transcription of bacteriophage T7 gene 10. Specifically, abortive transcripts produced from promoter phi 10 exert trans-acting antitermination activity on terminator T phi both in vitro and in vivo. Following abortive initiation cycling of T7 RNA polymerase at phi 10, short G-rich and oligo(G) RNAs were produced and both specifically sequestered 5- and 6-nt C + U stretch sequences, consequently interfering with terminator hairpin formation. This antitermination activity depended on sequence-specific hybridization of abortive transcripts with the 5' but not 3' half of T phi RNA. Antitermination was abolished when T phi was mutated to lack a C + U stretch, but restored when abortive transcript sequence was additionally modified to complement the mutation in T phi, both in vitro and in vivo. Antitermination was enhanced in vivo when the abortive transcript concentration was increased via overproduction of RNA polymerase or ribonuclease deficiency. Accordingly, antitermination activity exerted on T phi by abortive transcripts should facilitate expression of T phi-downstream promoter-less genes 11 and 12 in T7 infection of Escherichia coli.

  20. Tumor suppressor genes are larger than apoptosis-effector genes and have more regions of active chromatin: Connection to a stochastic paradigm for sequential gene expression programs.

    PubMed

    Garcia, Marlene; Mauro, James A; Ramsamooj, Michael; Blanck, George

    2015-08-03

    Apoptosis- and proliferation-effector genes are substantially regulated by the same transactivators, with E2F-1 and Oct-1 being notable examples. The larger proliferation-effector genes have more binding sites for the transactivators that regulate both sets of genes, and proliferation-effector genes have more regions of active chromatin, i.e, DNase I hypersensitive and histone 3, lysine-4 trimethylation sites. Thus, the size differences between the 2 classes of genes suggest a transcriptional regulation paradigm whereby the accumulation of transcription factors that regulate both sets of genes, merely as an aspect of stochastic behavior, accumulate first on the larger proliferation-effector gene "traps," and then accumulate on the apoptosis effector genes, thereby effecting sequential activation of the 2 different gene sets. As IRF-1 and p53 levels increase, tumor suppressor proteins are first activated, followed by the activation of apoptosis-effector genes, for example during S-phase pausing for DNA repair. Tumor suppressor genes are larger than apoptosis-effector genes and have more IRF-1 and p53 binding sites, thereby likewise suggesting a paradigm for transcription sequencing based on stochastic interactions of transcription factors with different gene classes. In this report, using the ENCODE database, we determined that tumor suppressor genes have a greater number of open chromatin regions and histone 3 lysine-4 trimethylation sites, consistent with the idea that a larger gene size can facilitate earlier transcriptional activation via the inclusion of more transactivator binding sites.

  1. PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a)

    PubMed Central

    Emerling, Brooke M.; Weinberg, Frank; Liu, Juinn-Lin; Mak, Tak W.; Chandel, Navdeep S.

    2008-01-01

    The tumor suppressor PTEN is mutated or deleted in many tumors, causing the activation of the PI3K pathway. Here, we show that the loss of PTEN increases the transcriptional activity of hypoxia-inducible factor 1 (HIF-1) through the inactivation of Forkhead transcription factors (FOXO) in PTEN-null cells. Reintroduction of PTEN into the nucleus, overexpression of a nonphosphorylatable FOXO3a, which accumulates in the nucleus, or inhibition of nuclear export of FOXO3a by leptomycin B represses HIF-1 transcriptional activity in PTEN-null cells. HIF-1 transcriptional activity increases in PTEN-positive cells depleted of FOXO3a with siRNA. PTEN and FOXO3a regulate the transactivation domain of HIF-1α. Chromatin immunoprecipitation indicates that FOXO3a complexes with HIF-1α and p300 on the Glut-1 promoter, a HIF-1 target gene. Overexpression of p300 reverses FOXO3a-mediated repression of HIF-1 transcriptional activity. Coimmunoprecipitation and GAL4-HIF-1α transactivation assays reveal that FOXO3a interferes with p300-dependent HIF-1 transcriptional activity. Thus, FOXO3a negatively regulates HIF-1 transcriptional activity. PMID:18268343

  2. Interaction of the Transcription Start Site Core Region and Transcription Factor YY1 Determine Ascorbate Transporter SVCT2 Exon 1a Promoter Activity

    PubMed Central

    Qiao, Huan; May, James M.

    2012-01-01

    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. PMID:22532872

  3. Differences in Transcriptional Activity of Human Papillomavirus Type 6 Molecular Variants in Recurrent Respiratory Papillomatosis

    PubMed Central

    Measso do Bonfim, Caroline; Simão Sobrinho, João; Lacerda Nogueira, Rodrigo; Salgado Kupper, Daniel; Cardoso Pereira Valera, Fabiana; Lacerda Nogueira, Maurício; Villa, Luisa Lina; Rahal, Paula; Sichero, Laura

    2015-01-01

    A significant proportion of recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus type 6 (HPV-6). The long control region (LCR) contains cis-elements for regulation of transcription. Our aim was to characterize LCR HPV-6 variants in RRP cases, compare promoter activity of these isolates and search for cellular transcription factors (TFs) that could explain the differences observed. The complete LCR from 13 RRP was analyzed. Transcriptional activity of 5 variants was compared using luciferase assays. Differences in putative TFs binding sites among variants were revealed using the TRANSFAC database. Chromatin immunoprecipation (CHIP) and luciferase assays were used to evaluate TF binding and impact upon transcription, respectively. Juvenile-onset RRP cases harbored exclusively HPV-6vc related variants, whereas among adult-onset cases HPV-6a variants were more prevalent. The HPV-6vc reference was more transcriptionally active than the HPV-6a reference. Active FOXA1, ELF1 and GATA1 binding sites overlap variable nucleotide positions among isolates and influenced LCR activity. Furthermore, our results support a crucial role for ELF1 on transcriptional downregulation. We identified TFs implicated in the regulation of HPV-6 early gene expression. Many of these factors are mutated in cancer or are putative cancer biomarkers, and must be further studied. PMID:26151558

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

    PubMed

    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

  5. Enhancer Activation Requires Trans-Recruitment of a Mega Transcription Factor Complex

    PubMed Central

    Liu, Zhijie; Merkurjev, Daria; Yang, Feng; Li, Wenbo; Oh, Soohwan; Friedman, Meyer J.; Song, Xiaoyuan; Zhang, Feng; Ma, Qi; Ohgi, Kenneth; Krones, Anna; Rosenfeld, Michael G.

    2014-01-01

    Summary Enhancers provide critical information directing cell-type specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here we report that the most strongly activated estrogen (E2)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome. PMID:25303530

  6. Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland.

    PubMed

    Villela, Darine; de Sá Lima, Larissa; Peres, Rafael; Peliciari-Garcia, Rodrigo Antonio; do Amaral, Fernanda Gaspar; Cipolla-Neto, José; Scavone, Cristóforo; Afeche, Solange Castro

    2014-01-17

    The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with β1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family.

    PubMed

    Schmidt, A; Vogel, R; Holloway, M K; Rutledge, S J; Friedman, O; Yang, Z; Rodan, G A; Friedman, E

    1999-09-10

    LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.

  9. Assessment of Anaerobic Toluene Biodegradation Activity by bssA Transcript/Gene Ratios

    PubMed Central

    Brow, Christina N.; O'Brien Johnson, Reid; Johnson, Richard L.

    2013-01-01

    Benzylsuccinate synthase (bssA) genes associated with toluene degradation were profiled across a groundwater contaminant plume under nitrate-reducing conditions and were detected in significant numbers throughout the plume. However, differences between groundwater and core sediment samples suggested that microbial transport, rather than local activity, was the underlying cause of the high copy numbers within the downgradient plume. Both gene transcript and reactant concentrations were consistent with this hypothesis. Expression of bssA genes from denitrifying toluene degraders was induced by toluene but only in the presence of nitrate, and transcript abundance dropped rapidly following the removal of either toluene or nitrate. The drop in bssA transcripts following the removal of toluene could be described by an exponential decay function with a half-life on the order of 1 h. Interestingly, bssA transcripts never disappeared completely but were always detected at some level if either inducer was present. Therefore, the detection of transcripts alone may not be sufficient evidence for contaminant degradation. To avoid mistakenly associating basal-level gene expression with actively degrading microbial populations, an integrated approach using the ratio of functional gene transcripts to gene copies is recommended. This approach minimizes the impact of microbial transport on activity assessment and allows reliable assessments of microbial activity to be obtained from water samples. PMID:23811506

  10. Regulation of gene expression by manipulating transcriptional repressor activity using a novel CoSRI technology.

    PubMed

    Xu, Yue; Li, Song Feng; Parish, Roger W

    2017-07-01

    Targeted gene manipulation is a central strategy for studying gene function and identifying related biological processes. However, a methodology for manipulating the regulatory motifs of transcription factors is lacking as these factors commonly possess multiple motifs (e.g. repression and activation motifs) which collaborate with each other to regulate multiple biological processes. We describe a novel approach designated conserved sequence-guided repressor inhibition (CoSRI) that can specifically reduce or abolish the repressive activities of transcription factors in vivo. The technology was evaluated using the chimeric MYB80-EAR transcription factor and subsequently the endogenous WUS transcription factor. The technology was employed to develop a reversible male sterility system applicable to hybrid seed production. In order to determine the capacity of the technology to regulate the activity of endogenous transcription factors, the WUS repressor was chosen. The WUS repression motif could be inhibited in vivo and the transformed plants exhibited the wus-1 phenotype. Consequently, the technology can be used to manipulate the activities of transcriptional repressor motifs regulating beneficial traits in crop plants and other eukaryotic organisms. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  11. The transcription factor p53: Not a repressor, solely an activator

    PubMed Central

    Fischer, Martin; Steiner, Lydia; Engeland, Kurt

    2014-01-01

    The predominant function of the tumor suppressor p53 is transcriptional regulation. It is generally accepted that p53-dependent transcriptional activation occurs by binding to a specific recognition site in promoters of target genes. Additionally, several models for p53-dependent transcriptional repression have been postulated. Here, we evaluate these models based on a computational meta-analysis of genome-wide data. Surprisingly, several major models of p53-dependent gene regulation are implausible. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and falsifies models of direct repression. This notion is supported by experimental re-analysis of representative genes reported as directly repressed by p53. Therefore, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors as well as models based on the function of ncRNAs are also not supported by the meta-analysis. As an alternative to models of direct repression, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21-DREAM/RB pathway. PMID:25486564

  12. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    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 facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  13. Transcriptional activation of human mu-opioid receptor gene by insulin-like growth factor-I in neuronal cells is modulated by the transcription factor REST.

    PubMed

    Bedini, Andrea; Baiula, Monica; Spampinato, Santi

    2008-06-01

    The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. We investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I up-regulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 signaling pathway and this transcription factor, binding to the signal transducer and activator of transcription-1/3 DNA element located in the promoter, increases OPRM1 transcription. We propose that a reduction in REST is a critical switch enabling IGF-I to up-regulate hMOPr. These findings help clarify how hMOPr expression is regulated in neuronal cells.

  14. CK1/Doubletime activity delays transcription activation in the circadian clock

    PubMed Central

    O'Neil, Jenna L; Merz, Gregory E; Dusad, Kritika; Crane, Brian R; Young, Michael W

    2018-01-01

    In the Drosophila circadian clock, Period (PER) and Timeless (TIM) proteins inhibit Clock-mediated transcription of per and tim genes until PER is degraded by Doubletime/CK1 (DBT)-mediated phosphorylation, establishing a negative feedback loop. Multiple regulatory delays within this feedback loop ensure ~24 hr periodicity. Of these delays, the mechanisms that regulate delayed PER degradation (and Clock reactivation) remain unclear. Here we show that phosphorylation of certain DBT target sites within a central region of PER affect PER inhibition of Clock and the stability of the PER/TIM complex. Our results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence. The role of DBT in regulating PER activity, stabilization and degradation ensures that these events are chronologically and biochemically linked, and contributes to the timing of an essential delay that influences the period of the circadian clock. PMID:29611807

  15. Hyperforin activates gene transcription involving transient receptor potential C6 channels.

    PubMed

    Thiel, Gerald; Rössler, Oliver G

    2017-04-01

    Hypericum perforatum is one of the most prominent medical plants. Hyperforin, a main ingredient of H. perforatum, has been shown to activate transient receptor potential canonical C6 (TRPC6) channels. Alternatively, it has been proposed that hyperforin functions as a protonophore in a TRPC6-independent manner. Here, we show that hyperforin stimulation activates the transcription factor AP-1 in HEK293 cells expressing TRPC6 (T6.11 cells), but did not substantially change the AP-1 activity in HEK293 cells lacking TRPC6. We identified the AP-1 binding site as a hyperforin-responsive element. AP-1 is composed of the transcription factors c-Jun and c-Fos, or other members of the c-Jun and c-Fos families of proteins. Hyperforin stimulation increased c-Jun and c-Fos promoter activities in T6.11 cells and induced an upregulation of c-Jun and c-Fos biosynthesis. The analysis of the c-Fos promoter revealed that the cAMP-response element also functions as a hyperforin-responsive element. Hyperforin-induced upregulation of AP-1 in T6.11 cells was attenuated by preincubation of the cells with either pregnenolone or progesterone, indicating that gene regulation via TRPC6 is under control of hormones or hormonal precursors. The signal transduction of hyperforin-induced AP-1 gene transcription required an influx of Ca 2+ ions into the cells, the activation of MAP kinases, and the activation of the transcription factors c-Jun and ternary complex factor. We conclude that hyperforin regulates gene transcription via activation of TRPC6 channels, involving stimulus-regulated protein kinases and stimulus-responsive transcription factors. The fact that hyperforin regulates gene transcription may explain many of the intracellular alterations induced by this compound. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. The Human RNA Polymerase I Transcription Terminator Complex Acts as a Replication Fork Barrier That Coordinates the Progress of Replication with rRNA Transcription Activity.

    PubMed

    Akamatsu, Yufuko; Kobayashi, Takehiko

    2015-05-01

    In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Distinct modes of gene regulation by a cell-specific transcriptional activator.

    PubMed

    Sengupta, Tanushri; Cohet, Nathalie; Morlé, François; Bieker, James J

    2009-03-17

    The architectural layout of a eukaryotic RNA polymerase II core promoter plays a role in general transcriptional activation. However, its role in tissue-specific expression is not known. For example, differing modes of its recognition by general transcription machinery can provide an additional layer of control within which a single tissue-restricted transcription factor may operate. Erythroid Kruppel-like factor (EKLF) is a hematopoietic-specific transcription factor that is critical for the activation of subset of erythroid genes. We find that EKLF interacts with TATA binding protein-associated factor 9 (TAF9), which leads to important consequences for expression of adult beta-globin. First, TAF9 functionally supports EKLF activity by enhancing its ability to activate the beta-globin gene. Second, TAF9 interacts with a conserved beta-globin downstream promoter element, and ablation of this interaction by beta-thalassemia-causing mutations decreases its promoter activity and disables superactivation. Third, depletion of EKLF prevents recruitment of TAF9 to the beta-globin promoter, whereas depletion of TAF9 drastically impairs beta-promoter activity. However, a TAF9-independent mode of EKLF transcriptional activation is exhibited by the alpha-hemoglobin-stabilizing protein (AHSP) gene, which does not contain a discernable downstream promoter element. In this case, TAF9 does not enhance EKLF activity and depletion of TAF9 has no effect on AHSP promoter activation. These studies demonstrate that EKLF directs different modes of tissue-specific transcriptional activation depending on the architecture of its target core promoter.

  18. DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways

    PubMed Central

    Mellström, Britt; Sahún, Ignasi; Ruiz-Nuño, Ana; Murtra, Patricia; Gomez-Villafuertes, Rosa; Savignac, Magali; Oliveros, Juan C.; Gonzalez, Paz; Kastanauskaite, Asta; Knafo, Shira; Zhuo, Min; Higuera-Matas, Alejandro; Errington, Michael L.; Maldonado, Rafael; DeFelipe, Javier; Jefferys, John G. R.; Bliss, Tim V. P.; Dierssen, Mara

    2014-01-01

    Changes in nuclear Ca2+ homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K+ channel interacting protein 3), is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory. PMID:24366545

  19. THRAP3 interacts with and inhibits the transcriptional activity of SOX9 during chondrogenesis.

    PubMed

    Sono, Takashi; Akiyama, Haruhiko; Miura, Shigenori; Deng, Jian Min; Shukunami, Chisa; Hiraki, Yuji; Tsushima, Yu; Azuma, Yoshiaki; Behringer, Richard R; Matsuda, Shuichi

    2018-07-01

    Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.

  20. A compendium of transcription factor and Transcriptionally active protein coding gene families in cowpea (Vigna unguiculata L.).

    PubMed

    Misra, Vikram A; Wang, Yu; Timko, Michael P

    2017-11-22

    Cowpea (Vigna unguiculata (L.) Walp.) is the most important food and forage legume in the semi-arid tropics of sub-Saharan Africa where approximately 80% of worldwide production takes place primarily on low-input, subsistence farm sites. Among the major goals of cowpea breeding and improvement programs are the rapid manipulation of agronomic traits for seed size and quality and improved resistance to abiotic and biotic stresses to enhance productivity. Knowing the suite of transcription factors (TFs) and transcriptionally active proteins (TAPs) that control various critical plant cellular processes would contribute tremendously to these improvement aims. We used a computational approach that employed three different predictive pipelines to data mine the cowpea genome and identified over 4400 genes representing 136 different TF and TAP families. We compare the information content of cowpea to two evolutionarily close species common bean (Phaseolus vulgaris), and soybean (Glycine max) to gauge the relative informational content. Our data indicate that correcting for genome size cowpea has fewer TF and TAP genes than common bean (4408 / 5291) and soybean (4408/ 11,065). Members of the GROWTH-REGULATING FACTOR (GRF) and Auxin/indole-3-acetic acid (Aux/IAA) gene families appear to be over-represented in the genome relative to common bean and soybean, whereas members of the MADS (Minichromosome maintenance deficient 1 (MCM1), AGAMOUS, DEFICIENS, and serum response factor (SRF)) and C2C2-YABBY appear to be under-represented. Analysis of the AP2-EREBP APETALA2-Ethylene Responsive Element Binding Protein (AP2-EREBP), NAC (NAM (no apical meristem), ATAF1, 2 (Arabidopsis transcription activation factor), CUC (cup-shaped cotyledon)), and WRKY families, known to be important in defense signaling, revealed changes and phylogenetic rearrangements relative to common bean and soybean that suggest these groups may have evolved different functions. The availability of detailed

  1. Bone morphogenetic protein 2 activates Smad6 gene transcription through bone-specific transcription factor Runx2.

    PubMed

    Wang, Qing; Wei, Xiaochao; Zhu, Tianhui; Zhang, Ming; Shen, Run; Xing, Lianping; O'Keefe, Regis J; Chen, Di

    2007-04-06

    BMP-2 plays an essential role in osteoblast and chondrocyte differentiation, but its signaling mechanism has not been fully defined. In the present studies, we investigated the mechanism through which BMP-2 activates the Smad6 gene. A -2006/+45 Smad6 promoter-luciferase construct was generated along with deletions and Runx2 binding site mutations to examine the role of Smad1 and Runx2 signaling following BMP-2 stimulation in osteoblasts. Transfection of Runx2 or treatment with BMP-2-stimulated promoter activity of the -2006/+45 and -1191/+45 reporters but not the -829/+45 and -374/+45 reporters. No Smad1/5 binding site is present in the -1191/-829 region of the Smad6 promoter. Mutation of the OSE2-a site (-1036/-1031) completely abolished the stimulatory effect of Runx2 as well as BMP-2 on the -2006/+45 and -1191/+45 Smad6 reporters. Gel shift and chromatin immunoprecipitation (ChIP) assays showed that Runx2 binds the OSE2-a element. ChIP assays demonstrated that Smad1 also interacts with the OSE2-a site at the Smad6 promoter through Runx2. The protein degradation of Runx2 is mediated by the E3 ubiquitin ligase Smurf1. In the present studies, we found that Smurf1 binds the OSE2-a site through Runx2 and inhibits Smad6 gene transcription. Treatment with BMP-2 and transfection of Smad1 abolished Smurf1 binding to the OSE2 site. These results show that Smad1 binding excludes Smurf1 interaction with the OSE2 site and promotes Smad6 gene transcription.

  2. Domain architecture of the p62 subunit from the human transcription/repair factor TFIIH deduced by limited proteolysis and mass spectrometry analysis.

    PubMed

    Jawhari, Anass; Boussert, Stéphanie; Lamour, Valérie; Atkinson, R Andrew; Kieffer, Bruno; Poch, Olivier; Potier, Noelle; van Dorsselaer, Alain; Moras, Dino; Poterszman, Arnaud

    2004-11-16

    TFIIH is a multiprotein complex that plays a central role in both transcription and DNA repair. The subunit p62 is a structural component of the TFIIH core that is known to interact with VP16, p53, Eralpha, and E2F1 in the context of activated transcription, as well as with the endonuclease XPG in DNA repair. We used limited proteolysis experiments coupled to mass spectrometry to define structural domains within the conserved N-terminal part of the molecule. The first domain identified resulted from spontaneous proteolysis and corresponds to residues 1-108. The second domain encompasses residues 186-240, and biophysical characterization by fluorescence studies and NMR analysis indicated that it is at least partially folded and thus may correspond to a structural entity. This module contains a region of high sequence conservation with an invariant FWxxPhiPhi motif (Phi representing either tyrosine or phenylalanine), which was also found in other protein families and could play a key role as a protein-protein recognition module within TFIIH. The approach used in this study is general and can be straightforwardly applied to other multidomain proteins and/or multiprotein assemblies.

  3. Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

    PubMed Central

    Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

    2016-01-01

    Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731

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

    PubMed

    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.

  5. Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

    PubMed

    Zhang, Qian; Giebler, Holli A; Isaacson, Marisa K; Nyborg, Jennifer K

    2015-01-01

    In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo. These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust

  6. Inhibition of transcriptional activity of c-JUN by SIRT1

    SciTech Connect

    Gao Zhanguo; Ye Jianping

    2008-11-28

    c-JUN is a major component of heterodimer transcription factor AP-1 (Activator Protein-1) that activates gene transcription in cell proliferation, inflammation and stress responses. SIRT1 (Sirtuin 1) is a histone deacetylase that controls gene transcription through modification of chromatin structure. However, it is not clear if SIRT1 regulates c-JUN activity in the control of gene transcription. Here, we show that SIRT1 associated with c-JUN in co-immunoprecipitation of whole cell lysate, and inhibited the transcriptional activity of c-JUN in the mammalian two hybridization system. SIRT1 was found in the AP-1 response element in the matrix metalloproteinase-9 (MMP9) promoter DNA leading to inhibitionmore » of histone 3 acetylation as shown in a ChIP assay. The SIRT1 signal was reduced by the AP-1 activator PMA, and induced by the SIRT1 activator Resveratrol in the promoter DNA. SIRT1-mediaetd inhibition of AP-1 was demonstrated in the MMP9 gene expression at the gene promoter, mRNA and protein levels. In mouse embryonic fibroblast (MEF) with SIRT1 deficiency (SIRT1{sup -/-}), mRNA and protein of MMP9 were increased in the basal condition, and the inhibitory activity of Resveratrol was significantly attenuated. Glucose-induced MMP9 expression was also inhibited by SIRT1 in response to Resveratrol. These data consistently suggest that SIRT1 directly inhibits the transcriptional activity of AP-1 by targeting c-JUN.« less

  7. Rational Design of Mini-Cas9 for Transcriptional Activation.

    PubMed

    Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

    2018-04-20

    Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

  8. Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements*

    PubMed Central

    Shan, Jixiu; Örd, Daima; Örd, Tõnis; Kilberg, Michael S.

    2009-01-01

    Protein limitation in vivo or amino acid deprivation of cells in culture causes a signal transduction cascade consisting of activation of the kinase GCN2 (general control nonderepressible 2), phosphorylation of eukaryotic initiation factor 2, and increased synthesis of activating transcription factor (ATF) 4 by a translational control mechanism. In a self-limiting transcriptional program, ATF4 transiently activates a wide range of downstream target genes involved in transport, cellular metabolism, and other cell functions. Simultaneous activation of other signal transduction pathways by amino acid deprivation led to the question of whether or not the increased abundance of ATF4 alone was sufficient to trigger the transcriptional control mechanisms. Using 293 cells that ectopically express ATF4 in a tetracycline-inducible manner showed that ATF4 target genes were activated in the absence of amino acid deprivation. Ectopic expression of ATF4 alone resulted in effective recruitment of the general transcription machinery, but some reduction in histone modification was observed. These data document that ATF4 alone is sufficient to trigger the amino acid-responsive transcriptional control program. However, the absolute amount of ectopic ATF4 required to achieve the same degree of transcriptional activation observed after amino acid limitation was greater, suggesting that other factors may serve to enhance ATF4 function. PMID:19509279

  9. PU.1 is a major transcriptional activator of the tumour suppressor gene LIMD1

    PubMed Central

    Foxler, Daniel E.; James, Victoria; Shelton, Samuel J.; Vallim, Thomas Q. de A.; Shaw, Peter E.; Sharp, Tyson V.

    2011-01-01

    LIMD1 is a tumour suppressor gene (TSG) down regulated in ∼80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1. PMID:21402070

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

    DTIC Science & Technology

    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

  11. Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

    PubMed Central

    Matamales, Miriam

    2012-01-01

    Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance. PMID:24327840

  12. Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

    PubMed

    Matamales, Miriam

    2012-12-19

    Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

  13. Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

    PubMed

    Matamales, Miriam

    2012-01-01

    Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

  14. Activation of RNA Polymerase III Transcription in Cells Transformed by Simian Virus 40

    PubMed Central

    Larminie, Christopher G. C.; Sutcliffe, Josephine E.; Tosh, Kerrie; Winter, Andrew G.; Felton-Edkins, Zoe A.; White, Robert J.

    1999-01-01

    RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses. PMID:10373542

  15. Aptazyme-embedded guide RNAs enable ligand-responsive genome editing and transcriptional activation

    PubMed Central

    Tang, Weixin; Hu, Johnny H.; Liu, David R.

    2017-01-01

    Programmable sequence-specific genome editing agents such as CRISPR-Cas9 have greatly advanced our ability to manipulate the human genome. Although canonical forms of genome-editing agents and programmable transcriptional regulators are constitutively active, precise temporal and spatial control over genome editing and transcriptional regulation activities would enable the more selective and potentially safer use of these powerful technologies. Here, by incorporating ligand-responsive self-cleaving catalytic RNAs (aptazymes) into guide RNAs, we developed a set of aptazyme-embedded guide RNAs that enable small molecule-controlled nuclease-mediated genome editing and small molecule-controlled base editing, as well as small molecule-dependent transcriptional activation in mammalian cells. PMID:28656978

  16. Selective Cooperation between Fatty Acid Binding Proteins and Peroxisome Proliferator-Activated Receptors in Regulating Transcription

    PubMed Central

    Tan, Nguan-Soon; Shaw, Natacha S.; Vinckenbosch, Nicolas; Liu, Peng; Yasmin, Rubina; Desvergne, Béatrice; Wahli, Walter; Noy, Noa

    2002-01-01

    Lipophilic compounds such as retinoic acid and long-chain fatty acids regulate gene transcription by activating nuclear receptors such as retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). These compounds also bind in cells to members of the family of intracellular lipid binding proteins, which includes cellular retinoic acid-binding proteins (CRABPs) and fatty acid binding proteins (FABPs). We previously reported that CRABP-II enhances the transcriptional activity of RAR by directly targeting retinoic acid to the receptor. Here, potential functional cooperation between FABPs and PPARs in regulating the transcriptional activities of their common ligands was investigated. We show that adipocyte FABP and keratinocyte FABP (A-FABP and K-FABP, respectively) selectively enhance the activities of PPARγ and PPARβ, respectively, and that these FABPs massively relocate to the nucleus in response to selective ligands for the PPAR isotype which they activate. We show further that A-FABP and K-FABP interact directly with PPARγ and PPARβ and that they do so in a receptor- and ligand-selective manner. Finally, the data demonstrate that the presence of high levels of K-FABP in keratinocytes is essential for PPARβ-mediated induction of differentiation of these cells. Taken together, the data establish that A-FABP and K-FABP govern the transcriptional activities of their ligands by targeting them to cognate PPARs in the nucleus, thereby enabling PPARs to exert their biological functions. PMID:12077340

  17. Transcriptional regulation of human papillomavirus type 18 P105 promoter by the co-activator CBP.

    PubMed

    Valencia-Hernández, Armando; Cuevas-Bennett, Christian; Garrido, Efraín

    2007-01-01

    Human papillomaviruses (HPVs) are the etiological agents of cervical cancer, with HPV-16 and 18 being the representative types of the higher risk group. The expression of the viral genes with transforming activity (E6 and E7) is controlled by the upstream regulatory region (URR), a segment of the viral genome that contains elements recognized by several transcription factors. We have analyzed the participation of the cellular co-activator CBP on the transcriptional regulation of the HPV-18 URR. We generated mutants and 5' end deletion constructs derived from the HPV-18 URR and evaluated their transcriptional activity performing transient co-transfection assays on C-33A cells with a plasmid that over-expresses the co-activator CBP. We also performed quantitative chromatin immunoprecipitation assays to analyze the participation of the co-activator CBP on the HPV-18 P105 promoter. Our results demonstrate that in C-33A cells CBP acts as a strong activator of the HPV-18 P105 promoter by a mechanism that depends on the integrity of the SP1-binding site, directly correlating with the acetylation of the histone H3 that is involved in nucleosomal stability. We propose a mechanism of regulation of the HPV-18 P105 promoter by the cellular co-activator CBP, recruited by the transcription factor SP1. (c) 2008 S. Karger AG, Basel

  18. O-GlcNAc transferase regulates transcriptional activity of human Oct4.

    PubMed

    Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

    2017-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Synchronous activation of gonadotropin-releasing hormone gene transcription and secretion by pulsatile kisspeptin stimulation

    PubMed Central

    Choe, Han Kyoung; Kim, Hee-Dae; Park, Sung Ho; Lee, Han-Woong; Park, Jae-Yong; Seong, Jae Young; Lightman, Stafford L.; Son, Gi Hoon; Kim, Kyungjin

    2013-01-01

    Pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH) is essential for pituitary gonadotrope function. Although the importance of pulsatile GnRH secretion has been recognized for several decades, the mechanisms underlying GnRH pulse generation in hypothalamic neural networks remain elusive. Here, we demonstrate the ultradian rhythm of GnRH gene transcription in single GnRH neurons using cultured hypothalamic slices prepared from transgenic mice expressing a GnRH promoter-driven destabilized luciferase reporter. Although GnRH promoter activity in each GnRH neuron exhibited an ultradian pattern of oscillations with a period of ∼10 h, GnRH neuronal cultures exhibited partially synchronized bursts of GnRH transcriptional activity at ∼2-h intervals. Surprisingly, pulsatile administration of kisspeptin, a potent GnRH secretagogue, evoked dramatic synchronous activation of GnRH gene transcription with robust stimulation of pulsatile GnRH secretion. We also addressed the issue of hierarchical interaction between the circadian and ultradian rhythms by using Bmal1-deficient mice with defective circadian clocks. The circadian molecular oscillator barely affected basal ultradian oscillation of GnRH transcription but was heavily involved in kisspeptin-evoked responses of GnRH neurons. In conclusion, we have clearly shown synchronous bursts of GnRH gene transcription in the hypothalamic GnRH neuronal population in association with episodic neurohormone secretion, thereby providing insight into GnRH pulse generation. PMID:23509283

  20. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    PubMed Central

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

    2015-01-01

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

  1. Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism1[OPEN

    PubMed Central

    Garapati, Prashanth; Feil, Regina; Lunn, John Edward; Van Dijck, Patrick; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-01-01

    Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1. PMID:26149570

  2. Negative Correlation between the Diffusion Coefficient and Transcriptional Activity of the Glucocorticoid Receptor.

    PubMed

    Mikuni, Shintaro; Yamamoto, Johtaro; Horio, Takashi; Kinjo, Masataka

    2017-08-25

    The glucocorticoid receptor (GR) is a transcription factor, which interacts with DNA and other cofactors to regulate gene transcription. Binding to other partners in the cell nucleus alters the diffusion properties of GR. Raster image correlation spectroscopy (RICS) was applied to quantitatively characterize the diffusion properties of EGFP labeled human GR (EGFP-hGR) and its mutants in the cell nucleus. RICS is an image correlation technique that evaluates the spatial distribution of the diffusion coefficient as a diffusion map. Interestingly, we observed that the averaged diffusion coefficient of EGFP-hGR strongly and negatively correlated with its transcriptional activities in comparison to that of EGFP-hGR wild type and mutants with various transcriptional activities. This result suggests that the decreasing of the diffusion coefficient of hGR was reflected in the high-affinity binding to DNA. Moreover, the hyper-phosphorylation of hGR can enhance the transcriptional activity by reduction of the interaction between the hGR and the nuclear corepressors.

  3. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    SciTech Connect

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8more » is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.« less

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

    PubMed

    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.

  5. Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity.

    PubMed

    Aapola, Ulla; Liiv, Ingrid; Peterson, Pärt

    2002-08-15

    DNMT3L is a regulator of imprint establishment of normally methylated maternal genomic sequences. DNMT3L shows high similarity to the de novo DNA methyltransferases, DNMT3A and DNMT3B, however, the amino acid residues needed for DNA cytosine methyltransferase activity have been lost from the DNMT3L protein sequence. Apart from methyltransferase activity, Dnmt3a and Dnmt3b serve as transcriptional repressors associating with histone deacetylase (HDAC) activity. Here we show that DNMT3L can also repress transcription by binding directly to HDAC1 protein. We have identified the PHD-like zinc finger of the ATRX domain as a main repression motif of DNMT3L, through which DNMT3L recruits the HDAC activity needed for transcriptional silencing. Furthermore, we show that DNMT3L protein contains an active nuclear localisation signal at amino acids 156-159. These results describe DNMT3L as a co-repressor protein and suggest that a transcriptionally repressed chromatin organisation through HDAC activity is needed for establishment of genomic imprints.

  6. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation.

    PubMed

    Kumar, Raj; Calhoun, William J

    2008-12-01

    Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR) is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade. Taken together, site-specific phosphorylation and related kinase pathways play an important role in the action of the GR, and more precise mechanistic information will lead to fuller understanding of the complex nature of gene regulation by the GR- and

  7. Model of transcriptional activation by MarA in Escherichia coli.

    PubMed

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

    2009-12-01

    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.

  8. Role of Signal Transducer and Activator of Transcription 3 in Neuronal Survival and Regeneration

    PubMed Central

    Dziennis, Suzan; Alkayed, Nabil J.

    2009-01-01

    Synopsis Signal Transducers and Activators of Transcription (STATs) comprise a family of transcription factors that mediate a wide variety of biological functions in the central and peripheral nervous systems. Injury to neural tissue induces STAT activation, and STATs are increasingly recognized for their role in neuronal survival. In this review, we discuss the role of STAT3 during neural development and following ischemic and traumatic injury in brain, spinal cord and peripheral nerves. We focus on STAT3 because of the expanding body of literature that investigates protective and regenerative effects of growth factors, hormones and cytokines that use STAT3 to mediate their effect, in part through transcriptional upregulation of neuroprotective and neurotrophic genes. Defining the endogenous molecular mechanisms that lead to neuroprotection by STAT3 after injury might identify novel therapeutic targets against acute neural tissue damage as well as chronic neurodegenerative disorders. PMID:19145989

  9. Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.

    PubMed

    Mills, Ian G; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E

    2005-07-18

    Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

  10. Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors

    PubMed Central

    Mills, Ian G.; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E.

    2005-01-01

    Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription. PMID:16027218

  11. Kojic acid biosynthesis in Aspergillus oryzae is regulated by a Zn(II)(2)Cys(6) transcriptional activator and induced by kojic acid at the transcriptional level.

    PubMed

    Marui, Junichiro; Yamane, Noriko; Ohashi-Kunihiro, Sumiko; Ando, Tomohiro; Terabayashi, Yasunobu; Sano, Motoaki; Ohashi, Shinichi; Ohshima, Eiji; Tachibana, Kuniharu; Higa, Yoshitaka; Nishimura, Marie; Koike, Hideaki; Machida, Masayuki

    2011-07-01

    A gene encoding the Zn(II)(2)Cys(6) transcriptional factor is clustered with two genes involved in biosynthesis of a secondary metabolite, kojic acid (KA), in Aspergillus oryzae. We determined that the gene was essential for KA production and the transcriptional activation of KA biosynthetic genes, which were triggered by the addition of KA. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

    PubMed

    Nezich, Catherine L; Wang, Chunxin; Fogel, Adam I; Youle, Richard J

    2015-08-03

    The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

  13. How to Select a Good Training-data Subset for Transcription: Submodular Active Selection for Sequences

    DTIC Science & Technology

    2009-01-01

    selection and uncertainty sampling signif- icantly. Index Terms: Transcription, labeling, submodularity, submod- ular selection, active learning , sequence...name of batch active learning , where a subset of data that is most informative and represen- tative of the whole is selected for labeling. Often...representative subset. Note that our Fisher ker- nel is over an unsupervised generative model, which enables us to bootstrap our active learning approach

  14. SUPPRESSION OF HIF-1α TRANSCRIPTIONAL ACTIVITY BY THE HIF PROLYL HYDROXYLASE EGLN1*

    PubMed Central

    To, Kenneth K. W.; Huang, L. Eric

    2005-01-01

    The cellular response to hypoxia is, at least in part, mediated by the transcriptional regulation of hypoxia-responsive genes involved in balancing the intracellular ATP production and consumption. Recent evidence suggests that the transcription factor, HIF-1α, functions as a master regulator of oxygen homeostasis by controlling a broad range of cellular events in hypoxia. In normoxia, HIF-1α is targeted for destruction via prolyl hydroxylation, an oxygen-dependent modification that signals for recognition by the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor (VHL). Three HIF prolyl hydroxylases (EGLN1, EGLN2, and EGLN3) have been identified in mammals, among which, EGLN1 and EGLN3, are hypoxia-inducible at their mRNA levels in a HIF-1α-dependent manner. In this study, we demonstrate that apart from promoting HIF-1α proteolysis in normoxia, EGLN1 specifically represses HIF-1α transcriptional activity in hypoxia. Ectopic expression of EGLN1 inhibited HIF-1α transcriptional activity without altering its protein levels in a VHL-deficient cell line, indicating a discrete activity of EGLN1 in transcriptional repression. Conversely, silencing of EGLN1 expression augmented HIF-1α transcriptional activity and its target gene expression in hypoxia. Hence, we propose that the accumulated EGLN1 in hypoxia acts as a negative-feedback mechanism to modulate HIF-1α target gene expression. Our finding also provides new insight into the pharmacological manipulation of the HIF prolyl hydroxylase for ischemic diseases. PMID:16157596

  15. Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II.

    PubMed

    Egyházi, E; Ossoinak, A; Lee, J M; Greenleaf, A L; Mäkelä, T P; Pigon, A

    1998-07-10

    The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (pol II) subunit is a target for extensive phosphorylation in vivo. Using in vitro kinase assays it was found that several different protein kinases can phosphorylate the CTD including the transcription factor IIH-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Hoeijmakers, and J. M. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phosphoCTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and pol II staining disappear from non-heat-shock genes concomitantly with the abolishment of transcriptional activity of these genes. In contrast, the actively transcribing heat-shock genes, manifested as chromosomal puff 5C on chromosome IV (IV-5C), stain intensely for phosphoCTD, but are devoid of CDK7. Furthermore, the staining of puff IV-5C with anti-PCTD antibodies was not detectably influenced by the TFIIH kinase and transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Following heat-shock treatment, the transcription of non-heat-shock genes was completely eliminated, while newly formed heat-shock gene transcripts emerged in a DRB-resistant manner. Thus, heat shock in these cells induces a rapid clearance of CDK7 from the non-heat-shock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together suggest the existence of heat-shock-specific CTD phosphorylation in living cells. This phosphorylation is resistant to DRB treatment, suggesting that not only phosphorylation but also transcription of heat-shock genes is DRB resistant and that CDK7 in heat shock cells is not associated with TFIIH.

  16. Transcriptional activation of short interspersed elements by DNA-damaging agents.

    PubMed

    Rudin, C M; Thompson, C B

    2001-01-01

    Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

  17. Multiple transcription factor codes activate epidermal wound–response genes in Drosophila

    PubMed Central

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

    2009-01-01

    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. PMID:19168633

  18. Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities.

    PubMed

    Nagase, Hiroyuki; Omae, Naoki; Omori, Akiko; Nakagawasai, Osamu; Tadano, Takeshi; Yokosuka, Akihito; Sashida, Yutaka; Mimaki, Yoshihiro; Yamakuni, Tohru; Ohizumi, Yasushi

    2005-12-02

    cAMP response element (CRE) transcription is dysregulated in neurodegenerative disorders in the central nervous system (CNS), including polyglutamine diseases. As the first step to find natural compounds with protective action against neurodegeneration in the CNS, we here examined whether six citrus flavonoids, namely nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin, stimulated CRE-dependent transcription and induced neurite outgrowth in PC12D cells. Among the compounds, nobiletin most potently enhanced CRE-dependent transcription and neurite outgrowth by activating ERK/MAP kinase-dependent signalling to increase CREB phosphorylation. The transcription and neurite outgrowth were stimulated by nobiletin in a concentration-dependent manner, with a strong correlation between them. Furthermore, a 11-day oral administration of nobiletin rescued impaired memory in olfactory-bulbectomized mice documented to be accompanied by a cholinergic neurodegeneration. These results suggest that nobiletin with the activity to improve impaired memory may become a potential leading compound for drug development for neurodegenerative disorders exhibiting the dysregulated CRE-dependent transcription.

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

    PubMed

    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. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  20. Stat1-Vitamin D Receptor Interactions Antagonize 1,25-Dihydroxyvitamin D Transcriptional Activity and Enhance Stat1-Mediated Transcription

    PubMed Central

    Vidal, Marcos; Ramana, Chilakamarti V.; Dusso, Adriana S.

    2002-01-01

    The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-γ and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-γ action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-γ/Stat1-induced transcription. This novel 1,25D-IFN-γ cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties. PMID:11909970

  1. Versatility of Cooperative Transcriptional Activation: A Thermodynamical Modeling Analysis for Greater-Than-Additive and Less-Than-Additive Effects

    PubMed Central

    Frank, Till D.; Carmody, Aimée M.; Kholodenko, Boris N.

    2012-01-01

    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-than-additive responses when

  2. TRANSCRIPTION FACTOR ACTIVATION FOLLOWING EXPOSURE OF AN INTACT LUNG PREPARATION TO METALLIC PARTICULATE MATTER

    EPA Science Inventory

    TRANSCRIPTION FACTOR ACTIVATION FOLLOWING EXPOSURE OF AN INTACT LUNG PREPARATION TO METALLIC PARTICULATE MATTER

    James M. Samet1,2, Robert Silbajoris1, Tony Huang1 and Ilona Jaspers3

    1Human Studies Division, National Health and Environmental Effects Research Laborato...

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

    PubMed

    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

    2016-04-07

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Alternative Polyadenylation Regulates CELF1/CUGBP1 Target Transcripts Following T Cell Activation

    PubMed Central

    Beisang, Daniel; Reilly, Cavan; Bohjanen, Paul R.

    2014-01-01

    Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3′ end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3′ untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3′ end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation PMID:25123787

  5. Regulation of muscle GLUT-4 transcription by AMP-activated protein kinase.

    PubMed

    Zheng, D; MacLean, P S; Pohnert, S C; Knight, J B; Olson, A L; Winder, W W; Dohm, G L

    2001-09-01

    Skeletal muscle GLUT-4 transcription in response to treatment with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMP-activated protein kinase (AMPK), was studied in rats and mice. The increase in GLUT-4 mRNA levels in response to a single subcutaneous injection of AICAR, peaked at 13 h in white and red quadriceps muscles but not in the soleus muscle. The mRNA level of chloramphenicol acyltransferase reporter gene which is driven by 1,154 or 895 bp of the human GLUT-4 proximal promoter was increased in AICAR-treated transgenic mice, demonstrating the transcriptional upregulation of the GLUT-4 gene by AICAR. However, this induction of transcription was not apparent with 730 bp of the promoter. In addition, nuclear extracts from AICAR-treated mice bound to the consensus sequence of myocyte enhancer factor-2 (from -473 to -464) to a greater extent than from saline-injected mice. Thus AMP-activated protein kinase activation by AICAR increases GLUT-4 transcription by a mechanism that requires response elements within 895 bp of human GLUT-4 proximal promoter and that may be cooperatively mediated by myocyte enhancer factor-2.

  6. Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription

    SciTech Connect

    Montgomery, K.F.; Tarr, P.I.; Bomsztyk, K.

    1991-08-01

    Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor {alpha} (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), the authors demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5{prime} flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-{kappa}B and AP-1. Gel mobility shiftmore » assays demonstrate that TNF, IL-1, or LPS induces activation of NF-{kappa}B-like DNA binding activity in HUVEC. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-{kappa}B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-{kappa}B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kB-like proteins but does inhibit ELAM-1 gene transcription. They conclude that PKC-independent activation of NF-{kappa}B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.« less

  7. Melatonin biosynthesis enzymes recruit WRKY transcription factors to regulate melatonin accumulation and transcriptional activity on W-box in cassava.

    PubMed

    Wei, Yunxie; Liu, Guoyin; Chang, Yanli; Lin, Daozhe; Reiter, Russel J; He, Chaozu; Shi, Haitao

    2018-03-12

    Melatonin is widely involved in growth, development, and stress responses in plants. Although the melatonin synthesis enzymes have been identified in various plants, their interacting proteins remain unknown. Herein, overexpression of tryptophan decarboxylase 2 (MeTDC2)-interacting proteins, N-acetylserotonin O-methyltransferase 2 (MeASMT2) interacting proteins, and N-acetylserotonin O-methyltransferase 3 (MeASMT3) in cassava leaf protoplasts resulted in more melatonin than when other enzymes were overexpressed. Through yeast two-hybrid, 14 MeTDC2-interacting proteins, 24 MeASMT2 interacting proteins, and 9 MeASMT3-interacting proteins were identified. Notably, we highlighted MeWRKY20 and MeWRKY75 as common interacting proteins of the 3 enzymes, as evidenced by yeast two-hybrid, and in vivo bimolecular fluorescence complementation (BiFC). Moreover, co-overexpression of MeTDC2/MeASMT2/3 with MeWRKY20/75 in cassava leaf protoplasts did not only activated the transcriptional activities of MeWRKY20 and MeWRKY75 on W-box, but also induced the effects of MeTDC2, MeASMT2/3 on endogenous melatonin levels. Taken together, 3 melatonin synthesis enzymes (MeTDC2, MeASMT2/3) interact with MeWRKY20/75 to form a protein complex in cassava. This information significantly extends the knowledge of the complex modulation of plant melatonin signaling. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Nardilysin promotes hepatocellular carcinoma through activation of signal transducer and activator of transcription 3.

    PubMed

    Kasai, Yosuke; Toriguchi, Kan; Hatano, Etsuro; Nishi, Kiyoto; Ohno, Mikiko; Yoh, Tomoaki; Fukuyama, Keita; Nishio, Takahiro; Okuno, Masayuki; Iwaisako, Keiko; Seo, Satoru; Taura, Kojiro; Kurokawa, Masato; Kunichika, Makoto; Uemoto, Shinji; Nishi, Eiichiro

    2017-05-01

    Nardilysin (NRDC) is a metalloendopeptidase of the M16 family. We previously showed that NRDC activates inflammatory cytokine signaling, including interleukin-6-signal transducer and activator of transcription 3 (STAT3) signaling. NRDC has been implicated in the promotion of breast, gastric and esophageal cancer, as well as the development of liver fibrosis. In this study, we investigated the role of NRDC in the promotion of hepatocellular carcinoma (HCC), both clinically and experimentally. We found that NRDC expression was upregulated threefold in HCC tissue compared to the adjacent non-tumor liver tissue, which was confirmed by immunohistochemistry and western blotting. We also found that high serum NRDC was associated with large tumor size (>3 cm, P = 0.016) and poor prognosis after hepatectomy (median survival time 32.0 vs 73.9 months, P = 0.003) in patients with hepatitis C (n = 120). Diethylnitrosamine-induced hepatocarcinogenesis was suppressed in heterozygous NRDC-deficient mice compared to their wild-type littermates. Gene silencing of NRDC with miRNA diminished the growth of Huh-7 and Hep3B spheroids in vitro. Notably, phosphorylation of STAT3 was decreased in NRDC-depleted Huh-7 spheroids compared to control spheroids. The effect of a STAT3 inhibitor (S3I-201) on the growth of Huh-7 spheroids was reduced in NRDC-depleted cells relative to controls. Our results show that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC promotes tumor growth through activation of STAT3. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  9. Direct activation of Shroom3 transcription by Pitx proteins drives epithelial morphogenesis in the developing gut

    PubMed Central

    Chung, Mei-I; Nascone-Yoder, Nanette M.; Grover, Stephanie A.; Drysdale, Thomas A.; Wallingford, John B.

    2010-01-01

    Individual cell shape changes are essential for epithelial morphogenesis. A transcriptional network for epithelial cell shape change is emerging in Drosophila, but this area remains largely unexplored in vertebrates. The distinction is important as so far, key downstream effectors of cell shape change in Drosophila appear not to be conserved. Rather, Shroom3 has emerged as a central effector of epithelial morphogenesis in vertebrates, driving both actin- and microtubule-based cell shape changes. To date, the morphogenetic role of Shroom3 has been explored only in the neural epithelium, so the broad expression of this gene raises two important questions: what are the requirements for Shroom3 in non-neural tissues and what factors control Shroom3 transcription? Here, we show in Xenopus that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut and that Shroom3 transcription in the gut requires the Pitx1 transcription factor. Moreover, we show that Pitx proteins directly activate Shroom3 transcription, and we identify Pitx-responsive regulatory elements in the genomic DNA upstream of Shroom3. Finally, we show that ectopic expression of Pitx proteins is sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrate new breadth to the requirements for Shroom3 in morphogenesis, and they also provide a cell-biological basis for the role of Pitx transcription factors in morphogenesis. More generally, these results provide a foundation for deciphering the transcriptional network that underlies epithelial cell shape change in developing vertebrates. PMID:20332151

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

    PubMed

    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.

  11. Advanced Glycated End-Products Affect HIF-Transcriptional Activity in Renal Cells

    PubMed Central

    Bondeva, Tzvetanka; Heinzig, Juliane; Ruhe, Carola

    2013-01-01

    Advanced glycated end-products (AGEs) are ligands of the receptor for AGEs and increase in diabetic disease. MAPK organizer 1 (Morg1) via its binding partner prolyl-hydroxylase domain (PHD)-3 presumably plays a role in the regulation of hypoxia-inducible factor (HIF)-1α and HIF-2α transcriptional activation. The purpose of this study was to analyze the influence of AGEs on Morg1 expression and its correlation to PHD3 activity and HIF-transcriptional activity in various renal cell types. The addition of glycated BSA (AGE-BSA) significantly up-regulated Morg1 mRNA levels in murine mesangial cells and down-regulated it in murine proximal tubular cells and differentiated podocytes. These effects were reversible when the cells were preincubated with a receptor for α-AGE antibody. AGE-BSA treatment induced a relocalization of the Morg1 cellular distribution compared with nonglycated control-BSA. Analysis of PHD3 activity demonstrated an elevated PHD3 enzymatic activity in murine mesangial cells but an inhibition in murine proximal tubular cells and podocytes after the addition of AGE-BSA. HIF-transcriptional activity was also affected by AGE-BSA treatment. Reporter gene assays and EMSAs showed that AGEs regulate HIF- transcriptional activity under nonhypoxic conditions in a cell type-specific manner. In proximal tubular cells, AGE-BSA stimulation elevated mainly HIF-1α transcriptional activity and to a lesser extent HIF-2α. We also detected an increased expression of the HIF-1α and the HIF-2α proteins in kidneys from Morg1 heterozygous (HZ) placebo mice compared with the Morg1 wild-type (WT) placebo-treated mice, and the HIF-1α protein expression in the Morg1 HZ streptozotocin-treated mice was significantly higher than the WT streptozotocin-treated mice. Analysis of isolated mesangial cells from Morg1 HZ (±) and WT mice showed an inhibited PHD3 activity and an increased HIF-transcriptional activity in cells with only one Morg1 allele. These findings are

  12. Information-dependent enrichment analysis reveals time-dependent transcriptional regulation of the estrogen pathway of toxicity.

    PubMed

    Pendse, Salil N; Maertens, Alexandra; Rosenberg, Michael; Roy, Dipanwita; Fasani, Rick A; Vantangoli, Marguerite M; Madnick, Samantha J; Boekelheide, Kim; Fornace, Albert J; Odwin, Shelly-Ann; Yager, James D; Hartung, Thomas; Andersen, Melvin E; McMullen, Patrick D

    2017-04-01

    The twenty-first century vision for toxicology involves a transition away from high-dose animal studies to in vitro and computational models (NRC in Toxicity testing in the 21st century: a vision and a strategy, The National Academies Press, Washington, DC, 2007). This transition requires mapping pathways of toxicity by understanding how in vitro systems respond to chemical perturbation. Uncovering transcription factors/signaling networks responsible for gene expression patterns is essential for defining pathways of toxicity, and ultimately, for determining the chemical modes of action through which a toxicant acts. Traditionally, transcription factor identification is achieved via chromatin immunoprecipitation studies and summarized by calculating which transcription factors are statistically associated with up- and downregulated genes. These lists are commonly determined via statistical or fold-change cutoffs, a procedure that is sensitive to statistical power and may not be as useful for determining transcription factor associations. To move away from an arbitrary statistical or fold-change-based cutoff, we developed, in the context of the Mapping the Human Toxome project, an enrichment paradigm called information-dependent enrichment analysis (IDEA) to guide identification of the transcription factor network. We used a test case of activation in MCF-7 cells by 17β estradiol (E2). Using this new approach, we established a time course for transcriptional and functional responses to E2. ERα and ERβ were associated with short-term transcriptional changes in response to E2. Sustained exposure led to recruitment of additional transcription factors and alteration of cell cycle machinery. TFAP2C and SOX2 were the transcription factors most highly correlated with dose. E2F7, E2F1, and Foxm1, which are involved in cell proliferation, were enriched only at 24 h. IDEA should be useful for identifying candidate pathways of toxicity. IDEA outperforms gene set enrichment

  13. Transcription Factors of Lotus: Regulation of Isoflavonoid Biosynthesis Requires Coordinated Changes in Transcription Factor Activity1[W][OA

    PubMed Central

    Shelton, Dale; Stranne, Maria; Mikkelsen, Lisbeth; Pakseresht, Nima; Welham, Tracey; Hiraka, Hideki; Tabata, Satoshi; Sato, Shusei; Paquette, Suzanne; Wang, Trevor L.; Martin, Cathie; Bailey, Paul

    2012-01-01

    Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors. PMID:22529285

  14. Conformational changes accompany activation of reovirus RNA-dependent RNA transcription

    PubMed Central

    Mendez, Israel I.; Weiner, Scott G.; She, Yi-Min; Yeager, Mark; Coombs, Kevin M.

    2009-01-01

    Many critical biologic processes involve dynamic interactions between proteins and nucleic acids. Such dynamic processes are often difficult to delineate by conventional static methods. For example, while a variety of nucleic acid polymerase structures have been determined at atomic resolution, the details of how some multi-protein transcriptase complexes actively produce mRNA, as well as conformational changes associated with activation of such complexes, remain poorly understood. The mammalian reovirus innermost capsid (core) manifests all enzymatic activities necessary to produce mRNA from each of the 10 encased double-stranded RNA genes. We used rapid freezing and electron cryo-microscopy to trap and visualize transcriptionally active reovirus core particles and compared them to inactive core images. Rod-like density centered within actively transcribing core spike channels was attributed to exiting nascent mRNA. Comparative radial density plots of active and inactive core particles identified several structural changes in both internal and external regions of the icosahedral core capsid. Inactive and transcriptionally active cores were partially digested with trypsin and identities of initial tryptic peptides determined by mass spectrometry. Differentially-digested peptides, which also suggest transcription-associated conformational changes, were placed within the known 3-dimensional structures of major core proteins. PMID:18321727

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

    PubMed

    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.

  16. A transcription activator-like effector induction system mediated by proteolysis

    PubMed Central

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

    2016-01-01

    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 due to their customizable DNA binding specificity. In this work we expand 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 following the induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator agnostic. PMID:26854666

  17. c-rel activates but v-rel suppresses transcription from kappa B sites.

    PubMed Central

    Inoue, J; Kerr, L D; Ransone, L J; Bengal, E; Hunter, T; Verma, I M

    1991-01-01

    We show that the product of the protooncogene c-rel is a constituent of an NF-kappa B-like complex that binds to the kappa B site originally identified in the enhancer of immunoglobulin kappa light chain gene. c-rel protein synthesized in bacteria binds to the kappa B site in a sequence-specific manner. The rel-kappa B complex can be disrupted by incubation with anti-rel antibodies. The rel protein can form oligomers. The c-rel protein can activate transcription from promoters containing kappa B sites; v-rel, on the other hand, suppresses the transcription of genes linked to kappa B sites. Thus, v-rel may interfere with the normal transcriptional machinery of the cell by acting as a dominant negative mutant. Images PMID:2023921

  18. Mitochondrial targeting of HIF-1α inhibits hypoxia-induced apoptosis independently of its transcriptional activity.

    PubMed

    Li, Hong-Sheng; Zhou, Yan-Ni; Li, Lu; Li, Sheng-Fu; Long, Dan; Chen, Xue-Lu; Zhang, Jia-Bi; Li, You-Ping; Feng, Li

    2018-04-25

    The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates adaptive responses to hypoxia by nuclear translocation and regulation of gene expression. Mitochondrial changes are critical for the adaptive response to hypoxia. However, the transcriptional and non-transcriptional mechanisms by which HIF-1α regulates mitochondria under hypoxia are poorly understood. Here, we examined the subcellular localization of HIF-1α in human cells and identified a small fraction of HIF-1α that translocated to the mitochondria after exposure to hypoxia or hypoxia-mimicking pharmacological agents. To probe the function of this HIF-1α population, we ectopically expressed a mitochondrial-targeted form of HIF-1α (mito-HIF-1α). Expression of mito-HIF-1α was sufficient to attenuate apoptosis induced by exposure to hypoxia or H 2 O 2 -induced oxidative stress. Moreover, mito-HIF-1α expression reduced the production of reactive oxygen species, the collapse of mitochondrial membrane potential, and the expression of mitochondrial DNA-encoded mRNA in response to hypoxia. However, these functions of mito-HIF-1α were independent of its conventional transcriptional activity. Finally, the livers of mice with CCl 4 -induced fibrosis showed a progressive increase in HIF-1α association with the mitochondria, indicating the clinical relevance of this finding. These data suggested that mitochondrial HIF-1α protects against apoptosis independently of its well-known role as a transcription factor. Copyright © 2018. Published by Elsevier Inc.

  19. Increased global transcription activity as a mechanism of replication stress in cancer

    PubMed Central

    Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M.; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

    2016-01-01

    Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer. PMID:27725641

  20. Increased global transcription activity as a mechanism of replication stress in cancer.

    PubMed

    Kotsantis, Panagiotis; Silva, Lara Marques; Irmscher, Sarah; Jones, Rebecca M; Folkes, Lisa; Gromak, Natalia; Petermann, Eva

    2016-10-11

    Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRAS V12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRAS V12 , elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

  1. Nitric Oxide Is a Signal for NNR-Mediated Transcription Activation in Paracoccus denitrificans

    PubMed Central

    Van Spanning, Rob J. M.; Houben, Edith; Reijnders, Willem N. M.; Spiro, Stephen; Westerhoff, Hans V.; Saunders, Neil

    1999-01-01

    By using the ′lacZ gene, the activities of the nirI, nirS, and norC promoters were assayed in the wild type and in NNR-deficient mutants of Paracoccus denitrificans grown under various growth conditions. In addition, induction profiles of the three promoters in response to the presence of various nitrogenous oxides were determined. Transcription from the three promoters required the absence of oxygen and the presence both of the transcriptional activator NNR and of nitric oxide. The activity of the nnr promoter itself was halved after the cells had been switched from aerobic respiration to denitrification. This response was apparently not a result of autoregulation or of regulation by FnrP, since the nnr promoter was as active in the wild-type strain as it was in NNR- or FnrP-deficient mutants. PMID:10383987

  2. Smad3 allostery links TGF-β receptor kinase activation to transcriptional control

    PubMed Central

    Qin, Bin Y.; Lam, Suvana S.; Correia, John J.; Lin, Kai

    2002-01-01

    Smad3 transduces the signals of TGF-βs, coupling transmembrane receptor kinase activation to transcriptional control. The membrane-associated molecule SARA (Smad Anchor for Receptor Activation) recruits Smad3 for phosphorylation by the receptor kinase. Upon phosphorylation, Smad3 dissociates from SARA and enters the nucleus, in which its transcriptional activity can be repressed by Ski. Here, we show that SARA and Ski recognize specifically the monomeric and trimeric forms of Smad3, respectively. Thus, trimerization of Smad3, induced by phosphorylation, simultaneously activates the TGF-β signal by driving Smad3 dissociation from SARA and sets up the negative feedback mechanism by Ski. Structural models of the Smad3/SARA/receptor kinase complex and Smad3/Ski complex provide insights into the molecular basis of regulation. PMID:12154125

  3. Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing.

    PubMed

    van Blokland, R; ten Lohuis, M; Meyer, P

    1997-12-01

    The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

  4. Inhibition of Rac1 activity induces G1/S phase arrest through the GSK3/cyclin D1 pathway in human cancer cells.

    PubMed

    Liu, Linna; Zhang, Hongmei; Shi, Lei; Zhang, Wenjuan; Yuan, Juanli; Chen, Xiang; Liu, Juanjuan; Zhang, Yan; Wang, Zhipeng

    2014-10-01

    Rac1 has been shown to regulate the cell cycle in cancer cells. Yet, the related mechanism remains unclear. Thus, the present study aimed to investigate the mechanism involved in the regulation of G1/S phase transition by Rac1 in cancer cells. Inhibition of Rac1 by inhibitor NSC23766 induced G1/S phase arrest and inhibited the proliferation of A431, SW480 and U2-OS cells. Suppression of GSK3 by shRNA partially rescued G1/S phase arrest and inhibition of proliferation. Incubation of cells with NSC23766 reduced p-AKT and inactivated p-GSK3α and p-GSK3β, increased p-cyclin D1 expression and decreased the level of cyclin D1 protein. Consequently, cyclin D1 targeting transcriptional factor E2F1 expression, which promotes G1 to S phase transition, was also reduced. In contrast, constitutive active Rac1 resulted in increased p-AKT and inactivated p-GSK3α and p-GSK3β, decreased p-cyclin D1 expression and enhanced levels of cyclin D1 and E2F1 expression. Moreover, suppression of GSK3 did not alter p-AKT or Rac1 activity, but decreased p-cyclin D1 and increased total cyclin D1 protein. However, neither Rac1 nor GSK3 inhibition altered cyclin D1 at the RNA level. Moreover, after inhibition of Rac1 or GSK3 following proteasome inhibitor MG132 treatment, cyclin D1 expression at the protein level remained constant, indicating that Rac1 and GSK3 may regulate cyclin D1 turnover through phosphorylation and degradation. Therefore, our findings suggest that inhibition of Rac1 induces cell cycle G1/S arrest in cancer cells by regulation of the GSK3/cyclin D1 pathway.

  5. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering.

    PubMed

    Ratman, Dariusz; Vanden Berghe, Wim; Dejager, Lien; Libert, Claude; Tavernier, Jan; Beck, Ilse M; De Bosscher, Karolien

    2013-11-05

    The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  6. Pleiotropic biological activities of alternatively spliced TMPRSS2/ERG fusion gene transcripts

    PubMed Central

    Wang, Jianghua; Cai, Yi; Yu, Wendong; Ren, Chengxi; Spencer, David M.; Ittmann, Michael

    2008-01-01

    TMPRSS2/ERG gene fusions are found in the majority of prostate cancers; however, there is significant heterogeneity in the 5′ region of the alternatively spliced fusion gene transcripts. We have found that there is also significant heterogeneity within the coding exons as well. There is variable inclusion of a 72-bp exon and other novel alternatively spliced isoforms. To assess the biological significance of these alternatively spliced transcripts, we expressed various transcripts in primary prostatic epithelial cells and in an immortalized prostatic epithelial cell line, PNT1a. The fusion gene transcripts promoted proliferation, invasion and motility with variable activities that depended on the structure of the 5′ region encoding the TMPRSS2/ERG fusion and the presence of the 72-bp exon. Cotransfection of different isoforms further enhanced biological activity, mimicking the situation in vivo, in which multiple isoforms are expressed. Finally, knockdown of the fusion gene in VCaP cells resulted in inhibition of proliferation in vitro and tumor progression in an in vivo orthotopic mice model. Our results indicate that TMPRSS2/ERG fusion isoforms have variable biological activities promoting tumor initiation and progression and are consistent with our previous clinical observations indicating that certain TMPRSS2/ERG fusion isoforms are significantly correlated with more aggressive disease. PMID:18922926

  7. HIPK2 modulates p53 activity towards pro-apoptotic transcription.

    PubMed

    Puca, Rosa; Nardinocchi, Lavinia; Sacchi, Ada; Rechavi, Gideon; Givol, David; D'Orazi, Gabriella

    2009-10-14

    Activation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells. Knockdown of HIPK2 inhibited both adriamycin-induced Ser46 phosphorylation and Lys382 acetylation in p53 protein; however, while combination of ADR and zinc restored Ser46 phosphorylation it did not recover Lys382 acetylation. Chromatin immunoprecipitation studies showed that HIPK2 was required in vivo for efficient p300/p53 co-recruitment onto apoptotic promoters and that both p53 modifications at Ser46 and Lys382 were necessary for p53 apoptotic transcription. Thus, p53Lys382 acetylation in HIPK2 knockdown as well as p53 apoptotic activity in response to drug could be rescued by p300 overexpression. Similar effect was obtained with the Sirt1-inhibitor nicotinamide. Interestingly trichostatin A (TSA), the inhibitor of histone deacetylase complexes (HDAC) did not have effect, suggesting that Sirt1 was the deacetylase involved in p53 deacetylation in HIPK2 knockdown. These results reveal a novel role for HIPK2 in activating p53 apoptotic transcription. Our results indicate that HIPK2 may regulate the balance between p53 acetylation and deacetylation, by stimulating on one hand co

  8. Cardiac preconditioning with sphingosine-1-phosphate requires activation of signal transducer and activator of transcription-3

    PubMed Central

    Kelly-Laubscher, Roisin F; King, Jonathan C; Hacking, Damian; Somers, Sarin; Hastie, Samantha; Stewart, Tessa; Imamdin, Aqeela; Maarman, Gerald; Pedretti, Sarah; Lecour, Sandrine

    2014-01-01

    Summary Aims Sphingosine-1-phosphate (S1P) is a cardioprotective agent. Signal transducer and activator of transcription 3 (STAT-3) is a key mediator of many cardioprotective agents. We aimed to explore whether STAT-3 is a key mediator in S1P-induced preconditioning. Methods Langendorff-perfused hearts from Wistar rats and wild-type or cardiomyocyte-specific STAT-3 knockout mice were pre-treated with S1P (10 nmol/l), with or without the STAT-3 pathway inhibitor AG490, before an ischaemia–reperfusion insult. Triphenyltetrazolium chloride and Evans blue staining were used for the determination of infarct size. Western blot analysis was carried out on the S1P pre-treated hearts for detection of cytosolic, nuclear and mitochondrial phosphorylated and total STAT-3 proteins. Results Pre-treatment with S1P decreased the infarct size in isolated rat (5 ± 3% vs control 26 ± 8%, p < 0.01) and wild-type mouse hearts (13 ± 1% vs control 33 ± 3%, p < 0.05). This protective effect was abolished in the rat hearts pre-treated with AG490 (30 ± 10%, p = ns vs control) and in the hearts from STAT-3 knockout mice (35 ± 4% vs control 30 ± 3%, p = ns). Levels of phosphorylated STAT-3 were significantly increased in both the nuclear (p < 0.05 vs control) and mitochondrial (p < 0.05 vs control) fractions in the S1P pre-treated hearts, but remained unchanged in the cytosolic fraction (p = ns vs control). Conclusion These novel results demonstrate that pharmacological preconditioning with S1P in the isolated heart is mediated by activation of mitochondrial and nuclear STAT-3, therefore suggesting that S1P may be a novel therapeutic target to modulate mitochondrial and nuclear function in cardiovascular disease in order to protect the heart against ischaemia–reperfusion. PMID:25000441

  9. Intramolecular control of transcriptional activity by the NK2-specific domain in NK-2 homeodomain proteins

    PubMed Central

    Watada, Hirotaka; Mirmira, Raghavendra G.; Kalamaras, Julie; German, Michael S.

    2000-01-01

    The developmentally important homeodomain transcription factors of the NK-2 class contain a highly conserved region, the NK2-specific domain (NK2-SD). The function of this domain, however, remains unknown. The primary structure of the NK2-SD suggests that it might function as an accessory DNA-binding domain or as a protein–protein interaction interface. To assess the possibility that the NK2-SD may contribute to DNA-binding specificity, we used a PCR-based approach to identify a consensus DNA-binding sequences for Nkx2.2, an NK-2 family member involved in pancreas and central nervous system development. The consensus sequence (TCTAAGTGAGCTT) is similar to the known binding sequences for other NK-2 homeodomain proteins, but we show that the NK2-SD does not contribute significantly to specific DNA binding to this sequence. To determine whether the NK2-SD contributes to transactivation, we used GAL4-Nkx2.2 fusion constructs to map a powerful transcriptional activation domain in the C-terminal region beyond the conserved NK2-SD. Interestingly, this C-terminal region functions as a transcriptional activator only in the absence of an intact NK2-SD. The NK2-SD also can mask transactivation from the paired homeodomain transcription factor Pax6, but it has no effect on transcription by itself. These results demonstrate that the NK2-SD functions as an intramolecular regulator of the C-terminal activation domain in Nkx2.2 and support a model in which interactions through the NK2-SD regulate the ability of NK-2-class proteins to activate specific genes during development. PMID:10944215

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

    PubMed

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

    PubMed

    Hildt, E; Saher, G; Bruss, V; Hofschneider, P H

    1996-11-01

    It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

  12. Mitochondrial deficiency impairs hypoxic induction of HIF-1 transcriptional activity and retards tumor growth

    PubMed Central

    Koido, Masaru; Haga, Naomi; Furuno, Aki; Tsukahara, Satomi; Sakurai, Junko; Tani, Yuri; Sato, Shigeo; Tomida, Akihiro

    2017-01-01

    Mitochondria can be involved in regulating cellular stress response to hypoxia and tumor growth, but little is known about that mechanistic relationship. Here, we show that mitochondrial deficiency severely retards tumor xenograft growth with impairing hypoxic induction of HIF-1 transcriptional activity. Using mtDNA-deficient ρ0 cells, we found that HIF-1 pathway activation was comparable in slow-growing ρ0 xenografts and rapid-growing parental xenografts. Interestingly, we found that ex vivo ρ0 cells derived from ρ0 xenografts exhibited slightly increased HIF-1α expression and modest HIF-1 pathway activation regardless of oxygen concentration. Surprisingly, ρ0 cells, as well as parental cells treated with oxidative phosphorylation inhibitors, were unable to boost HIF-1 transcriptional activity during hypoxia, although HIF-1α protein levels were ordinarily increased in these cells under hypoxic conditions. These findings indicate that mitochondrial deficiency causes loss of hypoxia-induced HIF-1 transcriptional activity and thereby might lead to a constitutive HIF-1 pathway activation as a cellular adaptation mechanism in tumor microenvironment. PMID:28060746

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

    PubMed

    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.

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

    SciTech Connect

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji

    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 amore » 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.« less

  15. Resveratrol Induces Growth Arrest and Apoptosis through Activation of FOXO Transcription Factors in Prostate Cancer Cells

    PubMed Central

    Chen, Qinghe; Ganapathy, Suthakar; Singh, Karan P.; Shankar, Sharmila; Srivastava, Rakesh K.

    2010-01-01

    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 apoptosis, and

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

    SciTech Connect

    Iordanskiy, Sergey; Van Duyne, Rachel; Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702

    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 lowermore » 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

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

    PubMed Central

    Thakore, Pratiksha I.; Gersbach, Charles A.

    2016-01-01

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

  18. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

    PubMed

    Malik, Sohail; Roeder, Robert G

    2010-11-01

    The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

  19. FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons

    PubMed Central

    Gilley, Jonathan; Coffer, Paul J.; Ham, Jonathan

    2003-01-01

    Developing sympathetic neurons die by apoptosis when deprived of NGF. BIM, a BH3-only member of the BCL-2 family, is induced after NGF withdrawal in these cells and contributes to NGF withdrawal–induced death. Here, we have investigated the involvement of the Forkhead box, class O (FOXO) subfamily of Forkhead transcription factors in the regulation of BIM expression by NGF. We find that overexpression of FOXO transcription factors induces BIM expression and promotes death of sympathetic neurons in a BIM-dependent manner. In addition, we find that FKHRL1 (FOXO3a) directly activates the bim promoter via two conserved FOXO binding sites and that mutation of these sites abolishes bim promoter activation after NGF withdrawal. Finally, we show that FOXO activity contributes to the NGF deprivation–induced death of sympathetic neurons. PMID:12913110

  20. Direct observation of frequency modulated transcription in single cells using light activation

    PubMed Central

    Larson, Daniel R; Fritzsch, Christoph; Sun, Liang; Meng, Xiuhau; Lawrence, David S; Singer, Robert H

    2013-01-01

    Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI: http://dx.doi.org/10.7554/eLife.00750.001 PMID:24069527

  1. Transforming growth factor β-activated kinase 1 transcriptionally suppresses hepatitis B virus replication.

    PubMed

    Pang, Jinke; Zhang, Geng; Lin, Yong; Xie, Zhanglian; Liu, Hongyan; Tang, Libo; Lu, Mengji; Yan, Ran; Guo, Haitao; Sun, Jian; Hou, Jinlin; Zhang, Xiaoyong

    2017-01-03

    Hepatitis B Virus (HBV) replication in hepatocytes is restricted by the host innate immune system and related intracellular signaling pathways. Transforming growth factor β-activated kinase 1 (TAK1) is a key mediator of toll-like receptors and pro-inflammatory cytokine signaling pathways. Here, we report that silencing or inhibition of endogenous TAK1 in hepatoma cell lines leads to an upregulation of HBV replication, transcription, and antigen expression. In contrast, overexpression of TAK1 significantly suppresses HBV replication, while an enzymatically inactive form of TAK1 exerts no effect. By screening TAK1-associated signaling pathways with inhibitors and siRNAs, we found that the MAPK-JNK pathway was involved in TAK1-mediated HBV suppression. Moreover, TAK1 knockdown or JNK pathway inhibition induced the expression of farnesoid X receptor α, a transcription factor that upregulates HBV transcription. Finally, ectopic expression of TAK1 in a HBV hydrodynamic injection mouse model resulted in lower levels of HBV DNA and antigens in both liver and serum. In conclusion, our data suggest that TAK1 inhibits HBV primarily at viral transcription level through activation of MAPK-JNK pathway, thus TAK1 represents an intrinsic host restriction factor for HBV replication in hepatocytes.

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

    ERIC Educational Resources Information Center

    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…

  3. Identification of Ind transcription activation and repression domains required for dorsoventral patterning of the CNS.

    PubMed

    Von Ohlen, Tonia L; Moses, Cade

    2009-07-01

    Specification of cell fates across the dorsoventral axis of the central nervous system in Drosophila involves the subdivision of the neuroectoderm into three domains that give rise to three columns of neural precursor cells called neuroblasts. Ventral nervous system defective (Vnd), intermediate neuroblasts defective (Ind) and muscle segment homeobox (Msh) are expressed in the three columns from ventral to dorsal, respectively. The products of these genes play multiple important roles in formation and specification of the embryonic nervous system. Ind, for example, is known to play roles in two important processes. First, Ind is essential for formation of neuroblasts conjunction with SoxB class transcription factors. Sox class transcription factors are known to specify neural stem cells in vertebrates. Second, Ind plays an important role in patterning the CNS in conjunction with, vnd and msh, which is also similar to how vertebrates pattern their neural tube. This work focuses two important aspects of Ind function. First, we used multiple approaches to identify and characterize specific domains within the protein that confer repressor or activator ability. Currently, little is known about the presence of activation or repression domains within Ind. Here, we show that transcriptional repression by Ind requires multiple conserved domains within the protein, and that Ind has a transcriptional activation domain. Specifically, we have identified a novel domain, the Pst domain, that has transcriptional repression ability and appears to act independent of interaction with the co-repressor Groucho. This domain is highly conserved among insect species, but is not found in vertebrate Gsh class homeodomain proteins. Second, we show that Ind can and does repress vnd expression, but does so in a stage specific manner. We conclude from this that the function of Ind in regulating vnd expression is one of refinement and maintenance of the dorsal border.

  4. A chromatin activity based chemoproteomic approach reveals a transcriptional repressome for gene-specific silencing

    PubMed Central

    Liu, Cui; Yu, Yanbao; Liu, Feng; Wei, Xin; Wrobel, John A.; Gunawardena, Harsha P.; Zhou, Li; Jin, Jian; Chen, Xian

    2015-01-01

    Immune cells develop endotoxin tolerance (ET) after prolonged stimulation. ET increases the level of a repression mark H3K9me2 in the transcriptional-silent chromatin specifically associated with pro-inflammatory genes. However, it is not clear what proteins are functionally involved in this process. Here we show that a novel chromatin activity based chemoproteomic (ChaC) approach can dissect the functional chromatin protein complexes that regulate ET-associated inflammation. Using UNC0638 that binds the enzymatically active H3K9-specific methyltransferase G9a/GLP, ChaC reveals that G9a is constitutively active at a G9a-dependent mega-dalton repressome in primary endotoxin-tolerant macrophages. G9a/GLP broadly impacts the ET-specific reprogramming of the histone code landscape, chromatin remodeling, and the activities of select transcription factors. We discover that the G9a-dependent epigenetic environment promotes the transcriptional repression activity of c-Myc for gene-specific co-regulation of chronic inflammation. ChaC may be also applicable to dissect other functional protein complexes in the context of phenotypic chromatin architectures. PMID:25502336

  5. Distortion in the spacer region of Pm during activation of middle transcription of phage Mu.

    PubMed Central

    Artsimovitch, I; Kahmeyer-Gabbe, M; Howe, M M

    1996-01-01

    Transcription from the middle promoter, Pm, of phage Mu is initiated by Escherichia coli RNA polymerase holoenzyme (E sigma 70; RNAP) and the phage-encoded activator, Mor. Point mutations in the spacer region between the -10 hexamer and the Mor binding site result in changes of promoter activity in vivo. These mutations are located at the junction between a rigid T-tract and adjacent, potentially deformable G + C-rich DNA segment, suggesting that deformation of the spacer region may play a role in the transcriptional activation of Pm. This prediction was tested by using dimethyl sulfate and potassium permanganate footprinting analyses. Helical distortion involving strand separation was detected at positions -32 to -34, close to the predicted interface between Mor and RNAP. Promoter mutants in which this distortion was not detected exhibited a lack of melting in the -12 to -1 region and reduced promoter activity in vivo. We propose that complexes containing the distortion represent stressed intermediates rather than stable open complexes and thus can be envisaged as a transition state in the kinetic pathway of Pm activation in which stored torsional energy could be used to facilitate melting around the transcription start point. Images Fig. 2 Fig. 3 Fig. 4 PMID:8790343

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

    PubMed

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

    2002-02-15

    The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A. Copyright 2002 Elsevier Science Ltd.

  7. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

    PubMed

    Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

    2013-02-28

    Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

  8. POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

    PubMed

    Lee, Dong-Kee; Suh, Dongchul; Edenberg, Howard J; Hur, Man-Wook

    2002-07-26

    The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

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

    SciTech Connect

    Ji, Suena; Park, Sang Yoon; Roth, Juergen

    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-L1more » 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.« less

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

    SciTech Connect

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

    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 formore » 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

  11. The flavonoid fisetin promotes osteoblasts differentiation through Runx2 transcriptional activity.

    PubMed

    Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Wittrant, Yohann; Coxam, Véronique

    2014-06-01

    Flavonoids represent a group of polyphenolic compounds commonly found in daily nutrition with proven health benefits. Among this group, the flavonol fisetin has been previously shown to protect bone by repressing osteoclast differentiation. In the present study, we investigated the role of fisetin in regulating osteoblasts physiology. In vivo mice treated with LPSs exhibited osteoporosis features associated with a dramatic repression of osteoblast marker expression. In this model, inhibition of osteocalcin and type I collagen alpha 1 transcription was partially countered by a daily consumption of fisetin. Interestingly, in vitro, fisetin promoted both osteoblast alkaline phosphatase activity and mineralization process. To decipher how fisetin may exert its positive effect on osteoblastogenesis, we analyzed its ability to control the runt-related transcription factor 2 (Runx2), a key organizer in developing and maturing osteoblasts. While fisetin did not impact Runx2 mRNA and protein levels, it upregulated its transcriptional activity. Actually, fisetin stimulated the luciferase activity of a reporter plasmid driven by the osteocalcin gene promoter that contains Runx2 binding sites and promoted the mRNA expression of osteocalcin and type I collagen alpha 1 targets. Bone sparing properties of fisetin also rely on its positive influence on osteoblast differentiation and activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation

    PubMed Central

    2010-01-01

    Background Appropriate control of mitochondrial function, morphology and biogenesis are crucial determinants of the general health of eukaryotic cells. It is therefore imperative that we understand the mechanisms that co-ordinate mitochondrial function with environmental signaling systems. The regulation of yeast mitochondrial function in response to nutritional change can be modulated by PKA activity. Unregulated PKA activity can lead to the production of mitochondria that are prone to the production of ROS, and an apoptotic form of cell death. Results We present evidence that mitochondria are sensitive to the level of cAMP/PKA signaling and can respond by modulating levels of respiratory activity or committing to self execution. The inappropriate activation of one of the yeast PKA catalytic subunits, Tpk3p, is sufficient to commit cells to an apoptotic death through transcriptional changes that promote the production of dysfunctional, ROS producing mitochondria. Our data implies that cAMP/PKA regulation of mitochondrial function that promotes apoptosis engages the function of multiple transcription factors, including HAP4, SOK2 and SCO1. Conclusions We propose that in yeast, as is the case in mammalian cells, mitochondrial function and biogenesis are controlled in response to environmental change by the concerted regulation of multiple transcription factors. The visualization of cAMP/TPK3 induced cell death within yeast colonies supports a model that PKA regulation plays a physiological role in coordinating respiratory function and cell death with nutritional status in budding yeast. PMID:21108829

  13. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons

    PubMed Central

    Wang, W.; Wildes, C. P.; Pattarabanjird, T.; Sanchez, M. I.; Glober, G.F.; Matthews, G. A.; Tye, K. M.; Ting, A. Y

    2017-01-01

    Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically-encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally-applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high-to-low calcium signal ratio of 10 after 10 minutes of stimulation. Channelrhodopsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium. PMID:28650461

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

    PubMed Central

    Ablain, Julien; Leiva, Magdalena; Peres, Laurent; Fonsart, Julien; Anthony, Elodie

    2013-01-01

    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. PMID:23509325

  15. Age-dependent regulation of ERF-VII transcription factor activity in Arabidopsis thaliana.

    PubMed

    Giuntoli, Beatrice; Shukla, Vinay; Maggiorelli, Federica; Giorgi, Federico M; Lombardi, Lara; Perata, Pierdomenico; Licausi, Francesco

    2017-10-01

    The Group VII Ethylene Responsive Factors (ERFs-VII) RAP2.2 and RAP2.12 have been mainly characterized with regard to their contribution as activators of fermentation in plants. However, transcriptional changes measured in conditions that stabilize these transcription factors exceed the mere activation of this biochemical pathway, implying additional roles performed by the ERF-VIIs in other processes. We evaluated gene expression in transgenic Arabidopsis lines expressing a stabilized form of RAP2.12, or hampered in ERF-VII activity, and identified genes affected by this transcriptional regulator and its homologs, including some involved in oxidative stress response, which are not universally induced under anaerobic conditions. The contribution of the ERF-VIIs in regulating this set of genes in response to chemically induced or submergence-stimulated mitochondria malfunctioning was found to depend on the plant developmental stage. A similar age-dependent mechanism also restrained ERF-VII activity upon the core-hypoxic genes, independently of the N-end rule pathway, which is accounted for the control of the anaerobic response. To conclude, this study shed new light on a dual role of ERF-VII proteins under submergence: as positive regulators of the hypoxic response and as repressors of oxidative-stress related genes, depending on the developmental stage at which plants are challenged by stress conditions. © 2017 John Wiley & Sons Ltd.

  16. DNA Recognition by a σ 54 Transcriptional Activator from Aquifex aeolicus

    DOE PAGES

    Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; ...

    2014-08-23

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

  17. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.

    PubMed

    Wang, Wenjing; Wildes, Craig P; Pattarabanjird, Tanyaporn; Sanchez, Mateo I; Glober, Gordon F; Matthews, Gillian A; Tye, Kay M; Ting, Alice Y

    2017-09-01

    Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

  18. ERalpha and ERbeta expression and transcriptional activity are differentially regulated by HDAC inhibitors

    PubMed Central

    Duong, Vanessa; Licznar, Anne; Margueron, Raphaël; Boulle, Nathalie; Busson, Muriel; Lacroix, Matthieu; Katzenellenbogen, Benita S.; Cavaillès, Vincent; Lazennec, Gwendal

    2006-01-01

    The proliferative action of ERα largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERβ displays tumor-suppressor properties, thus supporting the interest to identify compounds which could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) up-regulated ERβ protein levels, whereas it decreased ERα expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERβ more strongly than that of ERα. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERβ expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERβ and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy. PMID:16158045

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

    PubMed

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

    2013-04-08

    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.

  20. Two transcriptional activators of N-acetylserotonin O-methyltransferase 2 and melatonin biosynthesis in cassava.

    PubMed

    Wei, Yunxie; Liu, Guoyin; Bai, Yujing; Xia, Feiyu; He, Chaozu; Shi, Haitao; Foyer, Christine

    2017-10-13

    Similar to the situation in animals, melatonin biosynthesis is regulated by four sequential enzymatic steps in plants. Although the melatonin synthesis genes have been identified in various plants, the upstream transcription factors of them remain unknown. In this study on cassava (Manihot esculenta), we found that MeWRKY79 and heat-shock transcription factor 20 (MeHsf20) targeted the W-box and the heat-stress elements (HSEs) in the promoter of N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively. The interaction between MeWRKY79, MeHsf20, and the MeASMT2 promoter was evidenced by the activation of promoter activity and chromatin immunoprecipitation (ChIP) in cassava protoplasts, and by an in vitro electrophoretic mobility shift assay (EMSA). The transcripts of MeWRKY79, MeHsf20, and MeASMT2 were all regulated by a 22-amino acid flagellin peptide (flg22) and by Xanthomonas axonopodis pv manihotis (Xam). In common with the phenotype of MeASMT2, transient expression of MeWRKY79 and MeHsf20 in Nicotiana benthamiana leaves conferred improved disease resistance. Through virus-induced gene silencing (VIGS) in cassava, we found that MeWRKY79- and MeHsf20-silenced plants showed lower transcripts of MeASMT2 and less accumulation of melatonin, which resulted in disease sensitivity that could be reversed by exogenous melatonin. Taken together, these results indicate that MeASMT2 is a target of MeWRKY79 and MeHsf20 in plant disease resistance. This study identifies novel upstream transcription factors of melatonin synthesis genes in cassava, thus extending our knowledge of the complex modulation of melatonin synthesis in plant defense. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer. PMID:26681117

  4. Transcriptional activation of mouse mast cell Protease-7 by activin and transforming growth factor-beta is inhibited by microphthalmia-associated transcription factor.

    PubMed

    Funaba, Masayuki; Ikeda, Teruo; Murakami, Masaru; Ogawa, Kenji; Tsuchida, Kunihiro; Sugino, Hiromu; Abe, Matanobu

    2003-12-26

    Previous studies have revealed that activin A and transforming growth factor-beta1 (TGF-beta1) induced migration and morphological changes toward differentiation in bone marrow-derived cultured mast cell progenitors (BMCMCs). Here we show up-regulation of mouse mast cell protease-7 (mMCP-7), which is expressed in differentiated mast cells, by activin A and TGF-beta1 in BMCMCs, and the molecular mechanism of the gene induction of mmcp-7. Smad3, a signal mediator of the activin/TGF-beta pathway, transcriptionally activated mmcp-7. Microphthalmia-associated transcription factor (MITF), a tissue-specific transcription factor predominantly expressed in mast cells, melanocytes, and heart and skeletal muscle, inhibited Smad3-mediated mmcp-7 transcription. MITF associated with Smad3, and the C terminus of MITF and the MH1 and linker region of Smad3 were required for this association. Complex formation between Smad3 and MITF was neither necessary nor sufficient for the inhibition of Smad3 signaling by MITF. MITF inhibited the transcriptional activation induced by the MH2 domain of Smad3. In addition, MITF-truncated N-terminal amino acids could associate with Smad3 but did not inhibit Smad3-mediated transcription. The level of Smad3 was decreased by co-expression of MITF but not of dominant-negative MITF, which resulted from proteasomal protein degradation. The changes in the level of Smad3 protein were paralleled by those in Smad3-mediated signaling activity. These findings suggest that MITF negatively regulates Smad-dependent activin/TGF-beta signaling in a tissue-specific manner.

  5. Transcriptional activity of TGFβ1 and its receptors genes in thyroid gland.

    PubMed

    Kajdaniuk, Dariusz; Marek, Anna; Marek, Bogdan; Mazurek, Urszula; Fila-Daniłow, Anna; Foltyn, Wanda; Morawiec-Szymonik, Elżbieta; Siemińśka, Lucyna; Nowak, Mariusz; Głogowska-Szeląg, Joanna; Niedziołka-Zielonka, Danuta; Seemann, Michał; Kos-Kudła, Beata

    2016-01-01

    Determination of gene-candidates' profile expression responsible for fibrosis, immunosuppression, angiogenesis, and neoplasia processes in the pathogenesis of thyroid gland disease. Sixty-three patients underwent thyroidectomy: 27 with non-toxic nodular goitre (NG), 22 with toxic nodular goitre (TNG), six with papillary cancer (PTC), and eight with Graves' disease (GD). In thyroid tissues, transcriptional activity of TGFbeta1 and its receptors TGFbetaRI, TGFbetaRII, and TGFbetaRIII genes were assessed using RT-qPCR (Reverse Transcriptase Quantitative Polymerase Chain Reaction). Molecular analysis was performed in tissues derived from GD and from the tumour centre (PTC, NG, TNG) and from peripheral parts of the removed lobe without histopathological lesions (tissue control). Control tissue for analysis performed in GD was an unchanged tissue derived from peripheral parts of the removed lobe of patients surgically treated for a single benign tumour. Strict regulation observed among transcriptional activity of TGFb1 and their receptor TGFbetaRI-III genes in control tissues is disturbed in all pathological tissues - it is completely disturbed in PTC and GD, and partially in NG and TNG. Additionally, higher transcriptional activity of TGFb1 gene in PTC in comparison with benign tissues (NG, GD) and lower expression of mRNA TGFbRII (than in TNG, GD) and mRNA TGFbetaRIII than in all studied benign tissues (NG, TNG, GD) suggests a pathogenetic importance of this cytokine and its receptors in PTC development. In GD tissue, higher transcriptional activity of TGFbetaRII and TGFbetaRIII genes as compared to other pathological tissues was observed, indicating a participation of the receptors in the pathomechanism of autoimmune thyroid disease (AITD). TGFbeta1 blood concentrations do not reflect pathological processes taking place in thyroid gland. (Endokrynol Pol 2016; 67 (4): 375-382).

  6. Structure, function, and tethering of DNA-binding domains in σ 54 transcriptional activators

    DOE PAGES

    Vidangos, Natasha; Maris, Ann E.; Young, Anisa; ...

    2013-07-02

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

  7. Diadenosine Tetraphosphate Hydrolase Is Part of the Transcriptional Regulation Network in Immunologically Activated Mast Cells▿

    PubMed Central

    Carmi-Levy, Irit; Yannay-Cohen, Nurit; Kay, Gillian; Razin, Ehud; Nechushtan, Hovav

    2008-01-01

    We previously discovered that microphthalmia transcription factor (MITF) and upstream stimulatory factor 2 (USF2) each forms a complex with its inhibitor histidine triad nucleotide-binding 1 (Hint-1) and with lysyl-tRNA synthetase (LysRS). Moreover, we showed that the dinucleotide diadenosine tetraphosphate (Ap4A), previously shown to be synthesized by LysRS, binds to Hint-1, and as a result the transcription factors are released from their suppression. Thus, transcriptional activity is regulated by Ap4A, suggesting that Ap4A is a second messenger in this context. For Ap4A to be unambiguously established as a second messenger, several criteria have to be fulfilled, including the presence of a metabolizing enzyme. Since several enzymes are able to hydrolize Ap4A, we provided here evidence that the “Nudix” type 2 gene product, Ap4A hydrolase, is responsible for Ap4A degradation following the immunological activation of mast cells. The knockdown of Ap4A hydrolase modulated Ap4A accumulation, resulting in changes in the expression of MITF and USF2 target genes. Moreover, our observations demonstrated that the involvement of Ap4A hydrolase in gene regulation is not a phenomenon exclusive to mast cells but can also be found in cardiac cells activated with the β-agonist isoproterenol. Thus, we have provided concrete evidence establishing Ap4A as a second messenger in the regulation of gene expression. PMID:18644867

  8. Diadenosine tetraphosphate hydrolase is part of the transcriptional regulation network in immunologically activated mast cells.

    PubMed

    Carmi-Levy, Irit; Yannay-Cohen, Nurit; Kay, Gillian; Razin, Ehud; Nechushtan, Hovav

    2008-09-01

    We previously discovered that microphthalmia transcription factor (MITF) and upstream stimulatory factor 2 (USF2) each forms a complex with its inhibitor histidine triad nucleotide-binding 1 (Hint-1) and with lysyl-tRNA synthetase (LysRS). Moreover, we showed that the dinucleotide diadenosine tetraphosphate (Ap(4)A), previously shown to be synthesized by LysRS, binds to Hint-1, and as a result the transcription factors are released from their suppression. Thus, transcriptional activity is regulated by Ap(4)A, suggesting that Ap(4)A is a second messenger in this context. For Ap(4)A to be unambiguously established as a second messenger, several criteria have to be fulfilled, including the presence of a metabolizing enzyme. Since several enzymes are able to hydrolyze Ap(4)A, we provided here evidence that the "Nudix" type 2 gene product, Ap(4)A hydrolase, is responsible for Ap(4)A degradation following the immunological activation of mast cells. The knockdown of Ap(4)A hydrolase modulated Ap(4)A accumulation, resulting in changes in the expression of MITF and USF2 target genes. Moreover, our observations demonstrated that the involvement of Ap(4)A hydrolase in gene regulation is not a phenomenon exclusive to mast cells but can also be found in cardiac cells activated with the beta-agonist isoproterenol. Thus, we have provided concrete evidence establishing Ap(4)A as a second messenger in the regulation of gene expression.

  9. Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants.

    PubMed

    Dollhofer, Veronika; Callaghan, Tony M; Griffith, Gareth W; Lebuhn, Michael; Bauer, Johann

    2017-07-01

    Bioaugmentation with anaerobic fungi (AF) is promising for improved biogas generation from lignocelluloses-rich substrates. However, before implementing AF into biogas processes it is necessary to investigate their natural occurrence, community structure and transcriptional activity in agricultural biogas plants. Thus, AF were detected with three specific PCR based methods: (i) Copies of their 18S genes were found in 7 of 10 biogas plants. (ii) Transcripts of a GH5 endoglucanase gene were present at low level in two digesters, indicating transcriptional cellulolytic activity of AF. (iii) Phylogeny of the AF-community was inferred with the 28S gene. A new Piromyces species was isolated from a PCR-positive digester. Evidence for AF was only found in biogas plants operated with high proportions of animal feces. Thus, AF were most likely transferred into digesters with animal derived substrates. Additionally, high process temperatures in combination with long retention times seemed to impede AF survival and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. The transcription factor Grainy head primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes.

    PubMed

    Jacobs, Jelle; Atkins, Mardelle; Davie, Kristofer; Imrichova, Hana; Romanelli, Lucia; Christiaens, Valerie; Hulselmans, Gert; Potier, Delphine; Wouters, Jasper; Taskiran, Ibrahim I; Paciello, Giulia; González-Blas, Carmen B; Koldere, Duygu; Aibar, Sara; Halder, Georg; Aerts, Stein

    2018-06-04

    Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, we found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. We show that deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. Our data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.

  11. A unique H2A histone variant occupies the transcriptional start site of active genes.

    PubMed

    Soboleva, Tatiana A; Nekrasov, Maxim; Pahwa, Anuj; Williams, Rohan; Huttley, Gavin A; Tremethick, David J

    2011-12-04

    Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at genes regulated during spermatogenesis.

  12. Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

    PubMed

    Jeong, Kwon; Kim, Kiyoon; Kim, Hunsung; Oh, Yoojung; Kim, Seong-Jin; Jo, Yunhee; Choe, Wonchae

    2015-06-01

    Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

  13. Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells

    PubMed Central

    JEONG, KWON; KIM, KIYOON; KIM, HUNSUNG; OH, YOOJUNG; KIM, SEONG-JIN; JO, YUNHEE; CHOE, WONCHAE

    2015-01-01

    Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions. PMID:26137159

  14. The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of σ54-Dependent Transcription

    PubMed Central

    2012-01-01

    Summary: Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ54 for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ54-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ54 to the bacterial cell and its unique role in regulating transcription. PMID:22933558

  15. Domain-specific c-Myc ubiquitylation controls c-Myc transcriptional and apoptotic activity

    PubMed Central

    Zhang, Qin; Spears, Erick; Boone, David N.; Li, Zhaoliang; Gregory, Mark A.; Hann, Stephen R.

    2013-01-01

    The oncogenic transcription factor c-Myc causes transformation and tumorigenesis, but it can also induce apoptotic cell death. Although tumor suppressors are necessary for c-Myc to induce apoptosis, the pathways and mechanisms are unclear. To further understand how c-Myc switches from an oncogenic protein to an apoptotic protein, we examined the mechanism of p53-independent c-Myc–induced apoptosis. We show that the tumor suppressor protein ARF mediates this switch by inhibiting ubiquitylation of the c-Myc transcriptional domain (TD). Whereas TD ubiquitylation is critical for c-Myc canonical transcriptional activity and transformation, inhibition of ubiquitylation leads to the induction of the noncanonical c-Myc target gene, Egr1, which is essential for efficient c-Myc–induced p53-independent apoptosis. ARF inhibits the interaction of c-Myc with the E3 ubiquitin ligase Skp2. Overexpression of Skp2, which occurs in many human tumors, inhibits the recruitment of ARF to the Egr1 promoter, leading to inhibition of c-Myc–induced apoptosis. Therapeutic strategies could be developed to activate this intrinsic apoptotic activity of c-Myc to inhibit tumorigenesis. PMID:23277542

  16. Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia.

    PubMed

    Bhosale, Priyanka G; Pandey, Manishkumar; Desai, Rajiv S; Patil, Asawari; Kane, Shubhada; Prabhash, Kumar; Mahimkar, Manoj B

    2016-11-01

    In the present study, we comprehensively analyzed the prevalence of transcriptionally active human papilloma virus (HPV) in tissue samples of Indian patients with leukoplakia, predominantly hyperplastic lesions and head and neck squamous cell carcinoma (HNSCC). In addition, saliva samples from patients with HNSCC were screened for HPV detection. P16 overexpression was analyzed by immunohistochemistry. Tissue samples of leukoplakia (n = 121) and HNSCC (n = 427) and saliva from patients with HNSCC (n = 215) were tested for HPV using nested polymerase chain reaction. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in situ hybridization. P16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, in saliva or tissue. E6/E7 mRNA positivity was observed in 8 HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx, with very little concordance between p16 overexpression and HPV positivity. All patients with HPV-positive saliva samples had transcriptionally active HPV present in their tumors. The presence of HPV DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV-associated tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Dimeric PROP1 binding to diverse palindromic TAAT sequences promotes its transcriptional activity.

    PubMed

    Nakayama, Michie; Kato, Takako; Susa, Takao; Sano, Akiko; Kitahara, Kousuke; Kato, Yukio

    2009-08-13

    Mutations in the Prop1 gene are responsible for murine Ames dwarfism and human combined pituitary hormone deficiency with hypogonadism. Recently, we reported that PROP1 is a possible transcription factor for gonadotropin subunit genes through plural cis-acting sites composed of AT-rich sequences containing a TAAT motif which differs from its consensus binding sequence known as PRDQ9 (TAATTGAATTA). This study aimed to verify the binding specificity and sequence of PROP1 by applying the method of SELEX (Systematic Evolution of Ligands by EXponential enrichment), EMSA (electrophoretic mobility shift assay) and transient transfection assay. SELEX, after 5, 7 and 9 generations of selection using a random sequence library, showed that nucleotides containing one or two TAAT motifs were accumulated and accounted for 98.5% at the 9th generation. Aligned sequences and EMSA demonstrated that PROP1 binds preferentially to 11 nucleotides composed of an inverted TAAT motif separated by 3 nucleotides with variation in the half site of palindromic TAAT motifs and with preferential requirement of T at the nucleotide number 5 immediately 3' to a TAAT motif. Transient transfection assay demonstrated first that dimeric binding of PROP1 to an inverted TAAT motif and its cognates resulted in transcriptional activation, whereas monomeric binding of PROP1 to a single TAAT motif and an inverted ATTA motif did not mediate activation. Thus, this study demonstrated that dimeric binding of PROP1 is able to recognize diverse palindromic TAAT sequences separated by 3 nucleotides and to exhibit its transcriptional activity.

  18. Recent behavioral history modifies coupling between cell activity and Arc gene transcription in hippocampal CA1 neurons.

    PubMed

    Guzowski, John F; Miyashita, Teiko; Chawla, Monica K; Sanderson, Jennifer; Maes, Levi I; Houston, Frank P; Lipa, Peter; McNaughton, Bruce L; Worley, Paul F; Barnes, Carol A

    2006-01-24

    The ability of neurons to alter their transcriptional programs in response to synaptic input is of fundamental importance to the neuroplastic mechanisms underlying learning and memory. Because of technical limitations of conventional gene detection methods, the current view of activity-dependent neural transcription derives from experiments in which neurons are assumed quiescent until a signaling stimulus is given. The present study was designed to move beyond this static model by examining how earlier episodes of neural activity influence transcription of the immediate-early gene Arc. Using a sensitive FISH method that detects primary transcript at genomic alleles, the proportion of hippocampal CA1 neurons that activate transcription of Arc RNA was constant at approximately 40% in response to both a single novel exploration session and daily sessions repeated over 9 days. This proportion is similar to the percentage of active neurons defined electrophysiologically. However, this close correspondence was disrupted in rats exposed briefly, but repeatedly, to the same environment within a single day. Arc transcription in CA1 neurons declined dramatically after as few as four 5-min sessions, despite stable electrophysiological activity during all sessions. Additional experiments indicate that the decrement in Arc transcription occurred at the cellular, rather than synaptic level, and was not simply linked to habituation to novelty. Thus, the neural genomic response is governed by recent, but not remote, cell firing history in the behaving animal. This state-dependence of neuronal transcriptional coupling provides a mechanism of metaplasticity and may regulate capacity for synaptic modification in neural networks.

  19. BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription

    SciTech Connect

    Wang Jian; Tan Juan; Zhang Xihui

    2010-05-10

    Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may bemore » responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription.« less

  20. BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription.

    PubMed

    Wang, Jian; Tan, Juan; Zhang, Xihui; Guo, Hongyan; Zhang, Qicheng; Guo, Tingting; Geng, Yunqi; Qiao, Wentao

    2010-05-10

    Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may be responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription. Copyright 2010 Elsevier Inc. All rights reserved.

  1. A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements.

    PubMed

    Henry, Kelli F; Kawashima, Tomokazu; Goldberg, Robert B

    2015-06-01

    Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.

  2. NF-{kappa}B p65 represses {beta}-catenin-activated transcription of cyclin D1

    SciTech Connect

    Hwang, Injoo; Choi, Yong Seok; Jeon, Mi-Ya

    2010-12-03

    Research highlights: {yields} Cyclin D1 transcription is directly activated by {beta}-catenin; however, {beta}-catenin-induced cyclin D1 transcription is reduced by NF-{kappa}B p65. {yields} Protein-protein interaction between NF-{kappa}B p65 and {beta}-catenin might be responsible for p65-mediated repression of cyclin D1. {yields} One of five putative binding sites, located further upstream of other sites, is the major {beta}-catenin binding site in the cyclin D1 promoter. {yields} NF-{kappa}B binding site in cyclin D1 is occupied not only by p65 but also by {beta}-catenin, which is dynamically regulated by the signal. -- Abstract: Signaling crosstalk between the {beta}-catenin and NF-{kappa}B pathways represents a functional network.more » To test whether the crosstalk also occurs on their common target genes, the cyclin D1 promoter was used as a model because it contains binding sites for both proteins. {beta}-catenin activated transcription from the cyclin D1 promoter, while co-expression of NF-{kappa}B p65 reduced {beta}-catenin-induced transcription. Chromatin immunoprecipitation revealed lithium chloride-induced binding of {beta}-catenin on one of the T-cell activating factor binding sites. More interestingly, {beta}-catenin binding was greatly reduced by NF-{kappa}B p65, possibly by the protein-protein interaction between the two proteins. Such a dynamic and complex binding of {beta}-catenin and NF-{kappa}B on promoters might contribute to the regulated expression of their target genes.« less

  3. A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

    DOE PAGES

    Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

    2015-03-22

    Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

  4. A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements

    SciTech Connect

    Henry, Kelli F.; Kawashima, Tomokazu; Goldberg, Robert B.

    Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5'-GAAAAGCGAA-3'), the 10 bp-like motif (5'-GAAAAACGAA-3'), and Region 2 motif (partial sequence 5'-TTGGT-3'). Here, we usemore » site-directed mutagenesis experiments in transgenic tobacco globularstage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5'-GAGTTA-3') and a third 10-bp-related sequence (5'-GAAAACCACA-3'). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. Lastly, a homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related bean species.« less

  5. Modulation of TEL transcription activity by interaction with the ubiquitin-conjugating enzyme UBC9

    PubMed Central

    Chakrabarti, Subhra Ranjan; Sood, Rashmi; Ganguly, Surajit; Bohlander, Stefan; Shen, Zhiyuan; Nucifora, Giuseppina

    1999-01-01

    The E-26 transforming specific (ETS)-related gene TEL, also known as ETV6, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The encoded protein contains two functional domains: a helix–loop–helix (HLH) domain (also known as pointed domain) located at the N terminus and a DNA-binding domain located at the C terminus. The HLH domain is involved in protein–protein interaction with itself and other members of the ETS family of transcription factors such as FLI1. TEL is a transcription factor, and we and others have shown that it is a repressor of gene expression. To understand further the role of TEL in the cell, we have used an in vivo interaction system to identify proteins that interact with TEL. We show that a protein, UBC9, interacts specifically with TEL in vitro and in vivo. UBC9 is a member of the family of ubiquitin-conjugating enzymes. These enzymes usually are involved in proteosome-mediated degradation; however, our data suggest that interaction of TEL with UBC9 does not lead to TEL degradation. Our studies show that UBC9 binds to TEL exclusively through the HLH domain of TEL. We also show that TEL expressed as fusion to the DNA-binding domain of Gal4 completely represses a Gal4-responsive promoter, but that the coexpression of UBC9 in the same system restores the activity of the promoter. Targeted point mutation of conserved amino acids in UBC9 essential for enzymatic ubiquitination of proteins does not affect interaction nor transcriptional activity. Based on our data, we conclude that UBC9 physically interacts with TEL through the HLH domain and that the interaction leads to modulation of the transcription activity of TEL. PMID:10377438

  6. Reciprocal Activation of Transcription Factors Underlies the Dichotomy between Proliferation and Invasion of Glioma Cells

    PubMed Central

    Dhruv, Harshil D.; McDonough Winslow, Wendy S.; Armstrong, Brock; Tuncali, Serdar; Eschbacher, Jenny; Kislin, Kerri; Loftus, Joseph C.; Tran, Nhan L.; Berens, Michael E.

    2013-01-01

    Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the “Go or Grow” hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the “Go or Grow” hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens in situ (n = 44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation in vitro, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration in vitro and invasion ex vivo. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These findings

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

    SciTech Connect

    Lau, Wen Min; Doucet, Michele; Huang, David

    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 thatmore » 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

  8. Transcriptional Regulation During Zygotic Genome Activation in Zebrafish and Other Anamniote Embryos.

    PubMed

    Wragg, J; Müller, F

    2016-01-01

    Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major cellular and molecular transitions generate a pluripotent cell mass. The activation of the zygotic genome occurs during the so-called maternal to zygotic transition and prepares the embryo for zygotic takeover from maternal factors, in the control of the development of cellular lineages during differentiation. Recent advances in next generation sequencing technologies have allowed the dissection of the genomic and epigenomic processes mediating this transition. These processes include reorganization of the chromatin structure to a transcriptionally permissive state, changes in composition and function of structural and regulatory DNA-binding proteins, and changeover of the transcriptome as it is overhauled from that deposited by the mother in the oocyte to a zygotically transcribed complement. Zygotic genome activation in zebrafish occurs 10 cell cycles after fertilization and provides an ideal experimental platform for elucidating the temporal sequence and dynamics of establishment of a transcriptionally active chromatin state and helps in identifying the determinants of transcription activation at polymerase II transcribed gene promoters. The relatively large number of pluripotent cells generated by the fast cell divisions before zygotic transcription provides sufficient biomass for next generation sequencing technology approaches to establish the temporal dynamics of events and suggest causative relationship between them. However, genomic and genetic technologies need to be improved further to capture the earliest events in development, where cell number is a limiting factor. These technologies need to be complemented with precise, inducible genetic interference studies using the latest genome editing tools to reveal the function of candidate determinants and to confirm the predictions made by classic

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

    PubMed

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

    2002-08-15

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

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

    PubMed Central

    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. 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 RafL613V, 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. PMID:25650441

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

    PubMed Central

    Stanley, Frederick M.; Linder, Kathryn M.; Cardozo, Timothy J.

    2015-01-01

    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. PMID:26379245

  12. A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

    PubMed Central

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

    2013-01-01

    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 one 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, respectively, qRT-PCR and Surveyor nuclease. The TALE toolbox described here will enable a broad range of biological applications. PMID:22222791

  13. Signal Transducers and Activators of Transcription (STAT) family members in helminth infections.

    PubMed

    Becerra-Díaz, Mireya; Valderrama-Carvajal, Héctor; Terrazas, Luis I

    2011-01-01

    Helminth parasites are a diverse group of multicellular organisms. Despite their heterogeneity, helminths share many common characteristics, such as the modulation of the immune system of their hosts towards a permissive state that favors their development. They induce strong Th2-like responses with high levels of IL-4, IL-5 and IL-13 cytokines, and decreased production of proinflammatory cytokines such as IFN-γ. IL-4, IFN-γ and other cytokines bind with their specific cytokine receptors to trigger an immediate signaling pathway in which different tyrosine kinases (e.g. Janus kinases) are involved. Furthermore, a seven-member family of transcription factors named Signal Transducers and Activators of Transcription (STAT) that initiate the transcriptional activation of different genes are also involved and regulate downstream the JAK/STAT signaling pathway. However, how helminths avoid and modulate immune responses remains unclear; moreover, information concerning STAT-mediated immune regulation during helminth infections is scarce. Here, we review the research on mice deficient in STAT molecules, highlighting the importance of the JAK/STAT signaling pathway in regulating susceptibility and/or resistance in these infections.

  14. Phosphorylation of measles virus nucleoprotein upregulates the transcriptional activity of minigenomic RNA.

    PubMed

    Hagiwara, Kyoji; Sato, Hiroki; Inoue, Yoshihisa; Watanabe, Akira; Yoneda, Misako; Ikeda, Fusako; Fujita, Kentaro; Fukuda, Hiroyuki; Takamura, Chizuko; Kozuka-Hata, Hiroko; Oyama, Masaaki; Sugano, Sumio; Ohmi, Shinobu; Kai, Chieko

    2008-05-01

    We report the first identification of phosphorylation sites of the nucleoprotein (N) of the family Paramyxoviridae. The N protein is known to be the most abundant protein in infected cells; it constructs the N-RNA complex (nucleocapsid) and supports transcription and replication of viral genomic RNA. To determine the role of phosphorylation of the N protein, we expressed the N protein of the HL strain of measles virus (MV) in mammalian cells and purified the nucleocapsid. After separation of the C-terminal region from the core region, phosphorylated amino acids were assayed using MALDI-TOF/TOF and ESI-Q-TOF MS analyses. Two amino acids, S479 and S510, were shown to be phosphorylated by both methods of analysis. Metabolic labeling of the N protein with (32)P demonstrated that these two sites are the major phosphorylated sites within the MV-N protein. In transcriptional analysis using negative-strand minigenomic RNA containing the ORF of the luciferase gene, mutants of each phosphorylation site showed approximately 80% reduction in luciferase activity compared with the wild-type N, suggesting that the phosphorylation of N protein is important in the activation of the transcription of viral mRNA and/or replication of the genome in vivo.

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

    SciTech Connect

    Kim, Nam Soo; Kim, Yoon-Jin; Cho, Si Young

    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. Putativemore » 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.« less

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

    PubMed

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

    SciTech Connect

    Anderson, Courtney M.; Hu, Jianxin; Thomas, Reuben

    2017-03-28

    Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by themore » SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.« less

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

    SciTech Connect

    Neff, Michael M.

    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.

  19. WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis[W

    PubMed Central

    Castrillo, Gabriel; Sánchez-Bermejo, Eduardo; de Lorenzo, Laura; Crevillén, Pedro; Fraile-Escanciano, Ana; TC, Mohan; Mouriz, Alfonso; Catarecha, Pablo; Sobrino-Plata, Juan; Olsson, Sanna; Leo del Puerto, Yolanda; Mateos, Isabel; Rojo, Enrique; Hernández, Luis E.; Jarillo, Jose A.; Piñeiro, Manuel; Paz-Ares, Javier; Leyva, Antonio

    2013-01-01

    Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing. PMID:23922208

  20. The Transcription Elongation Complex Directs Activation-Induced Cytidine Deaminase-Mediated DNA Deamination†

    PubMed Central

    Besmer, Eva; Market, Eleonora; Papavasiliou, F. Nina

    2006-01-01

    Activation-induced cytidine deaminase (AID) is a single-stranded DNA deaminase required for somatic hypermutation of immunoglobulin (Ig) genes, a key process in the development of adaptive immunity. Transcription provides a single-stranded DNA substrate for AID, both in vivo and in vitro. We present here an assay which can faithfully replicate all of the molecular features of the initiation of hypermutation of Ig genes in vivo. In this assay, which detects AID-mediated deamination in the context of transcription by Escherichia coli RNA polymerase, deamination targets either strand and declines in efficiency as the distance from the promoter increases. We show that AID binds DNA exposed by the transcribing polymerase, implicating the polymerase itself as the vehicle which distributes AID on DNA as it moves away from the promoter. PMID:16705187

  1. TRANSCRIPTIONAL INHIBITION OF INTERLEUKIN-12 PROMOTER ACTIVITY IN LEISHMANIA SPP.-INFECTED MACROPHAGES

    PubMed Central

    Jayakumar, Asha; Widenmaier, Robyn; Ma, Xiaojing; McDowell, Mary Ann

    2009-01-01

    To establish and persist within a host, Leishmania spp. parasites delay the onset of cell-mediated immunity by suppressing interleukin-12 (IL-12) production from host macrophages. Although it is established that Leishmania spp.-infected macrophages have impaired IL-12 production, the mechanisms that account for this suppression remain to be completely elucidated. Using a luciferase reporter assay assessing IL-12 transcription, we report here that Leishmania major, Leishmania donovani, and Leishmania chagasi inhibit IL-12 transcription in response to interferon-gamma, lipopolysaccharide, and CD40 ligand and that Leishmania spp. lipophosphoglycan, phosphoglycans, and major surface protein are not necessary for inhibition. In addition, all the Leishmania spp. strains and life-cycle stages tested inhibited IL-12 promoter activity. Our data further reveal that autocrine-acting host factors play no role in the inhibitory response and that phagocytosis signaling is necessary for inhibition of IL-12. PMID:18372625

  2. Conserved developmental alternative splicing of muscleblind-like (MBNL) transcripts regulates MBNL localization and activity.

    PubMed

    Terenzi, Fulvia; Ladd, Andrea N

    2010-01-01

    Muscleblind-like (MBNL) proteins have been shown to regulate pre-mRNA alternative splicing, and MBNL1 has been implicated in regulating fetal-to-adult transitions in alternative splicing in the heart. MBNL1 is highly conserved, exhibiting more than 95% identity at the amino acid level between birds and mammals. To investigate MBNL1 expression during embryonic heart development, we examined MBNL1 transcript and protein expression in the embryonic chicken heart from the formation of the primitive heart tube through cardiac morphogenesis (embryonic days 1.5 through 8). MBNL1 transcript levels remained steady throughout these stages, whereas MBNL1 protein levels increased and exhibited a shift in isoforms. MBNL1 has several alternatively spliced exons. Using RT-PCR, we determined that the inclusion of one of these, exon 5, decreases dramatically during cardiac morphogenesis. This developmental transition is conserved in mice. Functional analyses of MBNL1 isoforms containing or lacking exon 5-encoded sequences revealed that exon 5 is important for the regulation of the subcellular localization, RNA binding affinity, and alternative splicing activity of MBNL1 proteins. A second MBNL protein, MBNL2, is also expressed in the embryonic heart. We found that MBNL2 exon 5, which is paralogous to MBNL1 exon 5, is similarly regulated during embryonic heart development. Analysis of MBNL1 and MBNL2 transcripts in several embryonic tissues in chicken and mouse indicate that exon 5 alternative splicing is highly conserved and tissue-specific. Thus, we propose that conserved developmental stage- and tissue-specific alternative splicing of MBNL transcripts is an important mechanism by which MBNL activity is regulated during embryonic development.

  3. Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit.

    PubMed

    Ho, Emily V; Klenotich, Stephanie J; McMurray, Matthew S; Dulawa, Stephanie C

    2016-01-01

    Anorexia nervosa (AN) is a complex eating disorder with severe dysregulation of appetitive behavior. The activity-based anorexia (ABA) paradigm is an animal model in which rodents exposed to both running wheels and scheduled feeding develop aspects of AN including paradoxical hypophagia, dramatic weight loss, and hyperactivity, while animals exposed to only one condition maintain normal body weight. Brain-derived neurotrophic factor (BDNF), an activity-dependent modulator of neuronal plasticity, is reduced in the serum of AN patients, and is a known regulator of feeding and weight maintenance. We assessed the effects of scheduled feeding, running wheel access, or both on the expression of BDNF transcripts within the mesocorticolimbic pathway. We also assessed the expression of neuronal cell adhesion molecule 1 (NCAM1) to explore the specificity of effects on BDNF within the mesocorticolimbic pathway. Scheduled feeding increased the levels of both transcripts in the hippocampus (HPC), increased NCAM1 mRNA expression in the ventral tegmental area (VTA), and decreased BDNF mRNA levels in the medial prefrontal cortex (mPFC). In addition, wheel running increased BDNF mRNA expression in the VTA. No changes in either transcript were observed in the nucleus accumbens (NAc). Furthermore, no changes in either transcript were induced by the combined scheduled feeding and wheel access condition. These data indicate that scheduled feeding or wheel running alter BDNF and NCAM1 expression levels in specific regions of the mesocorticolimbic pathway. These findings contribute to our current knowledge of the molecular alterations induced by ABA and may help elucidate possible mechanisms of AN pathology.

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

    SciTech Connect

    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 themore » 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 m

  5. Enhanced cellulase production in Trichoderma reesei RUT C30 via constitution of minimal transcriptional activators.

    PubMed

    Zhang, Jiajia; Zhang, Guoxiu; Wang, Wei; Wang, Wei; Wei, Dongzhi

    2018-05-17

    Cellulase can convert lignocellulosic feedstocks into fermentable sugars, which can be used for the industrial production of biofuels and chemicals. The high cost of cellulase production remains a challenge for lignocellulose breakdown. Trichoderma reesei RUT C30 serves as a well-known industrial workhorse for cellulase production. Therefore, the enhancement of cellulase production by T. reesei RUT C30 is of great importance. Two sets of novel minimal transcriptional activators (DBD ace2 -VP16 and DBD cre1 -VP16) were designed and expressed in T. reesei RUT C30. Expression of DBD ace2 -VP16 and DBD cre1 -VP16 improved cellulase production under induction (avicel or lactose) and repression (glucose) conditions, respectively. The strain T MTA66 under avicel and T MTA139 under glucose with the highest cellulase activities outperformed other transformants and the parental strain under the corresponding conditions. For T MTA66 strains, the highest FPase activity was approximately 1.3-fold greater than that of the parental strain RUT C30 at 120 h of cultivation in a shake flask using avicel as the sole carbon source. The FPase activity (U/mg biomass) in T MTA139 strains was approximately 26.5-fold higher than that of the parental strain RUT C30 at 72 h of cultivation in a shake flask using glucose as the sole carbon source. Furthermore, the crude enzymes produced in the 7-L fermenter from T MTA66 and T MTA139 supplemented with commercial β-glucosidase hydrolyzed pretreated corn stover effectively. These results show that replacing natural transcription factors with minimal transcriptional activators is a powerful strategy to enhance cellulase production in T. reesei. Our current study also offers an alternative genetic engineering strategy for the enhanced production of industrial products by other fungi.

  6. Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.

    PubMed

    Chen, M; Hieng, S; Qian, X; Costa, R; Ou, J H

    1994-11-15

    Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.

  7. Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

    PubMed

    Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

    2014-11-07

    Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and

  8. Mammalian transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes and are predicted to act as transcriptional activator hubs.

    PubMed

    Joshi, Anagha

    2014-12-30

    Transcriptional hotspots are defined as genomic regions bound by multiple factors. They have been identified recently as cell type specific enhancers regulating developmentally essential genes in many species such as worm, fly and humans. The in-depth analysis of hotspots across multiple cell types in same species still remains to be explored and can bring new biological insights. We therefore collected 108 transcription-related factor (TF) ChIP sequencing data sets in ten murine cell types and classified the peaks in each cell type in three groups according to binding occupancy as singletons (low-occupancy), combinatorials (mid-occupancy) and hotspots (high-occupancy). The peaks in the three groups clustered largely according to the occupancy, suggesting priming of genomic loci for mid occupancy irrespective of cell type. We then characterized hotspots for diverse structural functional properties. The genes neighbouring hotspots had a small overlap with hotspot genes in other cell types and were highly enriched for cell type specific function. Hotspots were enriched for sequence motifs of key TFs in that cell type and more than 90% of hotspots were occupied by pioneering factors. Though we did not find any sequence signature in the three groups, the H3K4me1 binding profile had bimodal peaks at hotspots, distinguishing hotspots from mono-modal H3K4me1 singletons. In ES cells, differentially expressed genes after perturbation of activators were enriched for hotspot genes suggesting hotspots primarily act as transcriptional activator hubs. Finally, we proposed that ES hotspots might be under control of SetDB1 and not DNMT for silencing. Transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes. In ES cells, they are predicted to act as transcriptional activator hubs and might be under SetDB1 control for silencing.

  9. Signal transducers and activators of transcription: STATs-mediated mitochondrial neuroprotection.

    PubMed

    Lin, Hung Wen; Thompson, John W; Morris, Kahlilia C; Perez-Pinzon, Miguel A

    2011-05-15

    Cerebral ischemia is defined as little or no blood flow in cerebral circulation, characterized by low tissue oxygen and glucose levels, which promotes neuronal mitochondria dysfunction leading to cell death. A strategy to counteract cerebral ischemia-induced neuronal cell death is ischemic preconditioning (IPC). IPC results in neuroprotection, which is conferred by a mild ischemic challenge prior to a normally lethal ischemic insult. Although many IPC-induced mechanisms have been described, many cellular and subcellular mechanisms remain undefined. Some reports have suggested key signal transduction pathways of IPC, such as activation of protein kinase C epsilon, mitogen-activated protein kinase, and hypoxia-inducible factors, that are likely involved in IPC-induced mitochondria mediated-neuroprotection. Moreover, recent findings suggest that signal transducers and activators of transcription (STATs), a family of transcription factors involved in many cellular activities, may be intimately involved in IPC-induced ischemic tolerance. In this review, we explore current signal transduction pathways involved in IPC-induced mitochondria mediated-neuroprotection, STAT activation in the mitochondria as it relates to IPC, and functional significance of STATs in cerebral ischemia.

  10. Signal Transducers and Activators of Transcription: STATs-Mediated Mitochondrial Neuroprotection

    PubMed Central

    Lin, Hung Wen; Thompson, John W.; Morris, Kahlilia C.

    2011-01-01

    Abstract Cerebral ischemia is defined as little or no blood flow in cerebral circulation, characterized by low tissue oxygen and glucose levels, which promotes neuronal mitochondria dysfunction leading to cell death. A strategy to counteract cerebral ischemia-induced neuronal cell death is ischemic preconditioning (IPC). IPC results in neuroprotection, which is conferred by a mild ischemic challenge prior to a normally lethal ischemic insult. Although many IPC-induced mechanisms have been described, many cellular and subcellular mechanisms remain undefined. Some reports have suggested key signal transduction pathways of IPC, such as activation of protein kinase C epsilon, mitogen-activated protein kinase, and hypoxia-inducible factors, that are likely involved in IPC-induced mitochondria mediated-neuroprotection. Moreover, recent findings suggest that signal transducers and activators of transcription (STATs), a family of transcription factors involved in many cellular activities, may be intimately involved in IPC-induced ischemic tolerance. In this review, we explore current signal transduction pathways involved in IPC-induced mitochondria mediated-neuroprotection, STAT activation in the mitochondria as it relates to IPC, and functional significance of STATs in cerebral ischemia. Antioxid. Redox Signal. 14, 1853–1861. PMID:20712401

  11. Erythropoietin activates telomerase through transcriptional and posttranscriptional regulation in human erythroleukemic JAS-REN-A cells.

    PubMed

    Akiyama, Masaharu; Kawano, Takeshi; Mikami-Terao, Yoko; Agawa-Ohta, Miyuki; Yamada, Osamu; Ida, Hiroyuki; Yamada, Hisashi

    2011-03-01

    We evaluated the molecular mechanism of telomerase activation by erythropoietin (EPO) in human erythroleukemic JAS-REN-A cells. Telomerase activity increased 3-4 fold after 3-24h of culture with EPO and was associated with increases in c-myc mRNA after 1-3h, of c-Myc protein after 3-6h, and of human telomerase reverse transcriptase (hTERT) mRNA and hTERT protein after 6-24h. Simultaneously EPO induced phosphorylation of signal transducer activator of transcription 5 (STAT5), AKT, and extracellular signal-regulated kinase (ERK). Telomerase activity induced by EPO was significantly inhibited by AG490, PD98059, and LY294002. AG490 downregulated c-myc and hTERT mRNA expression with inhibited STAT5 and AKT phosphorylation. PD98059 also reduced c-myc and hTERT expression and inhibited ERK phosphorylation. However, LY294002 did not inhibit c-myc or hTERT mRNA expression despite inhibiting STAT5 and AKT phosphorylation. These results suggest that EPO activates telomerase in JAS-REN-A cells through dual regulation: hTERT gene transcription by Janus tyrosine kinase 2/STAT5/c-Myc and hTERT protein phosphorylation by phosphatidylinositol 3'-kinase/AKT. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

    SciTech Connect

    Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel

    2007-09-21

    The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount ofmore » AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment.« less

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

    PubMed

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

    2016-07-01

    The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations

    PubMed Central

    Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

    2017-01-01

    Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators. PMID:28301511

  15. Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations.

    PubMed

    Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

    2017-01-01

    Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.

  16. Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

    SciTech Connect

    Jensen, Taylor J.; Wozniak, Ryan J.; Arizona Cancer Center, University of Arizona, Tucson, AZ 85724

    2009-02-15

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modificationsmore » and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.« less

  17. Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

    PubMed Central

    2014-01-01

    Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. PMID:24997498

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

    PubMed

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

    2000-06-01

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

  19. Jasmonate Regulates Plant Responses to Postsubmergence Reoxygenation through Transcriptional Activation of Antioxidant Synthesis1

    PubMed Central

    Dai, Yang-Shuo; Xie, Li-Juan; Yu, Lu-Jun; Zhou, Ying; Lai, Yong-Xia; Yang, Yi-Cong; Xu, Le; Chen, Qin-Fang

    2017-01-01

    Submergence induces hypoxia in plants; exposure to oxygen following submergence, termed reoxygenation, produces a burst of reactive oxygen species. The mechanisms of hypoxia sensing and signaling in plants have been well studied, but how plants respond to reoxygenation remains unclear. Here, we show that reoxygenation in Arabidopsis (Arabidopsis thaliana) involves rapid accumulation of jasmonates (JAs) and increased transcript levels of JA biosynthesis genes. Application of exogenous methyl jasmonate improved tolerance to reoxygenation in wild-type Arabidopsis; also, mutants deficient in JA biosynthesis and signaling were very sensitive to reoxygenation. Moreover, overexpression of the transcription factor gene MYC2 enhanced tolerance to posthypoxic stress, and myc2 knockout mutants showed increased sensitivity to reoxygenation, indicating that MYC2 functions as a key regulator in the JA-mediated reoxygenation response. MYC2 transcriptionally activates members of the VITAMIN C DEFECTIVE (VTC) and GLUTATHIONE SYNTHETASE (GSH) gene families, which encode rate-limiting enzymes in the ascorbate and glutathione synthesis pathways. Overexpression of VTC1 and GSH1 in the myc2-2 mutant suppressed the posthypoxic hypersensitive phenotype. The JA-inducible accumulation of antioxidants may alleviate oxidative damage caused by reoxygenation, improving plant survival after submergence. Taken together, our findings demonstrate that JA signaling interacts with the antioxidant pathway to regulate reoxygenation responses in Arabidopsis. PMID:28082717

  20. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration.

    PubMed

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-11-15

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk.

  1. Extrachromosomal HPV-16 LCR transcriptional activation by HDACi opposed by cellular differentiation and DNA integration

    PubMed Central

    Bojilova, Ekaterina Dimitrova; Weyn, Christine; Antoine, Marie-Hélène; Fontaine, Véronique

    2016-01-01

    Histone deacetylase inhibitors (HDACi) have been shown to render HPV-carrying cells susceptible to intrinsic and extrinsic apoptotic signals. As such, these epigenetic drugs have entered clinical trials in the effort to treat cervical cancer. Here, we studied the effect of common HDACi, with an emphasis on Trichostatin A (TSA), on the transcriptional activity of the HPV-16 Long Control Region (LCR) in order to better understand the impact of these agents in the context of the HPV life cycle and infection. HDACi strongly induced transcription of the firefly luciferase reporter gene under the control of the HPV-16 LCR in a variety of cell lines. In the HaCaT keratinocyte cell line undergoing differentiation induced by TSA, we observed a reduction in LCR-controlled transcription. Three major AP-1 binding sites in the HPV-16 LCR are involved in the regulation by TSA. However, whatever the status of differentiation of the HaCaT cells, TSA induced integration of extra-chromosomal transfected DNA into the cellular genome. Although these data suggest caution using HDACi in the treatment of HR HPV infection, further in vivo studies are necessary to better assess the risk. PMID:27705914

  2. Protective upregulation of activating transcription factor-3 against glutamate neurotoxicity in neuronal cells under ischemia.

    PubMed

    Takarada, Takeshi; Kou, Miki; Hida, Miho; Fukumori, Ryo; Nakamura, Saki; Kutsukake, Takaya; Kuramoto, Nobuyuki; Hinoi, Eiichi; Yoneda, Yukio

    2016-05-01

    This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs. © 2016 Wiley Periodicals, Inc.

  3. ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator.

    PubMed

    Jahn, Arne; Rane, Grishma; Paszkowski-Rogacz, Maciej; Sayols, Sergi; Bluhm, Alina; Han, Chung-Ting; Draškovič, Irena; Londoño-Vallejo, José Arturo; Kumar, Alan Prem; Buchholz, Frank; Butter, Falk; Kappei, Dennis

    2017-06-01

    Telomeres constitute the ends of linear chromosomes and together with the shelterin complex form a structure essential for genome maintenance and stability. In addition to the constitutive binding of the shelterin complex, other direct, yet more transient interactions are mediated by the CST complex and HOT1/HMBOX1, while subtelomeric variant repeats are recognized by NR2C/F transcription factors. Recently, the Kruppel-like zinc finger protein ZBTB48/HKR3/TZAP has been described as a novel telomere-associated factor in the vertebrate lineage. Here, we show that ZBTB48 binds directly both to telomeric and to subtelomeric variant repeat sequences. ZBTB48 is found at telomeres of human cancer cells regardless of the mode of telomere maintenance and it acts as a negative regulator of telomere length. In addition to its telomeric function, we demonstrate through a combination of RNAseq, ChIPseq and expression proteomics experiments that ZBTB48 acts as a transcriptional activator on a small set of target genes, including mitochondrial fission process 1 (MTFP1). This discovery places ZBTB48 at the interface of telomere length regulation, transcriptional control and mitochondrial metabolism. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  4. The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

    PubMed

    Kokabu, Shoichiro; Nakatomi, Chihiro; Matsubara, Takuma; Ono, Yusuke; Addison, William N; Lowery, Jonathan W; Urata, Mariko; Hudnall, Aaron M; Hitomi, Suzuro; Nakatomi, Mitsushiro; Sato, Tsuyoshi; Osawa, Kenji; Yoda, Tetsuya; Rosen, Vicki; Jimi, Eijiro

    2017-08-04

    Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Activation of Stat3 Transcription Factor by Herpesvirus Saimiri STP-A Oncoprotein

    PubMed Central

    Chung, Young-Hwa; Cho, Nam-hyuk; Garcia, Maria Ines; Lee, Sun-Hwa; Feng, Pinghui; Jung, Jae U.

    2004-01-01

    The saimiri transforming protein (STP) oncogene of Herpesvirus saimiri subgroup A strain 11 (STP-A11) is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. We previously showed that STP-A11 interacts with cellular Src kinase through its SH2 binding motif and that this interaction elicits Src signal transduction. Here we demonstrate that STP-A11 interacts with signal transducer and activator of transcription 3 (Stat3) independently of Src association and that the amino-terminal short proline-rich motif of STP-A11 and the central linker region of Stat3 are necessary for their interaction. STP-A11 formed a triple complex with Src kinase and Stat3 where Src kinase phosphorylated Stat3, resulting in the nuclear localization and transcriptional activation of Stat3. Consequently, the constitutively active Stat3 induced by STP-A11 elicited cellular signal transduction, which ultimately induced cell survival and proliferation upon serum deprivation. Furthermore, this activity was strongly correlated with the induction of Fos, cyclin D1, and Bcl-XL expression. These results demonstrate that STP-A11 independently targets two important cellular signaling molecules, Src and Stat3, and that these proteins cooperate efficiently to induce STP-A11-mediated transformation. PMID:15163742

  6. Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

    PubMed Central

    Hosogane, Masaki; Funayama, Ryo; Nishida, Yuichiro; Nagashima, Takeshi; Nakayama, Keiko

    2013-01-01

    Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity. PMID:24009517

  7. Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal.

    PubMed

    Pal, Shaifali; Yadav, Akhilesh Kumar; Singh, Anup Kumar; Rastogi, Shubhra; Gupta, Madan Mohan; Verma, Rajesh Kumar; Nagegowda, Dinesh A; Pal, Anirban; Shasany, Ajit Kumar

    2017-01-01

    The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

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

    PubMed

    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. Copyright © 2015 by the Genetics Society of America.

  9. Activity of the Rhodopseudomonas palustris p-coumaroyl-homoserine lactone-responsive transcription factor RpaR.

    PubMed

    Hirakawa, Hidetada; Oda, Yasuhiro; Phattarasukol, Somsak; Armour, Christopher D; Castle, John C; Raymond, Christopher K; Lappala, Colin R; Schaefer, Amy L; Harwood, Caroline S; Greenberg, E Peter

    2011-05-01

    The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.

  10. A homolog of teleostean signal transducer and activator of transcription 3 (STAT3) from rock bream, Oplegnathus fasciatus: Structural insights, transcriptional modulation, and subcellular localization.

    PubMed

    Bathige, S D N K; Thulasitha, William Shanthakumar; Umasuthan, Navaneethaiyer; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

    2017-04-01

    Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors in the Janus kinase (JAK)/STAT signaling pathway, and it was previously considered as acute phase response factor. A number of interleukins (ILs) such as IL-5, IL-6, IL-9, IL-10, IL-12, and IL-22 are known to be involved in activation of STAT3. In addition, various growth factors and pathogenic or oxidative stresses mediate the activation of a wide range of functions via STAT3. In this study, a STAT3 homolog was identified and functionally characterized from rock bream (RbSTAT3), Oplegnathus fasciatus. In silico characterization revealed that the RbSTAT3 amino acid sequence shares highly conserved common domain architectural features including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, and Src homology 2 (SH2) domains. In addition, a fairly conserved transcriptional activation domain (TAD) was located at the C-terminus. Comparison of RbSTAT3 with other counterparts revealed higher identities (>90%) with fish orthologs. The genomic sequence of RbSTAT3 was obtained from a bacterial artificial chromosome (BAC) library, and was identified as a multi-exonic gene (24 exons), as found in other vertebrates. Genomic structural comparison and phylogenetic studies have showed that the evolutionary routes of teleostean and non-teleostean vertebrates were distinct. Quantitative real time PCR (qPCR) analysis revealed that the spatial distribution of RbSTAT3 mRNA expression was ubiquitous and highly detectable in blood, heart, and liver tissues. Transcriptional modulation of RbSTAT3 was examined in blood and liver tissues after challenges with bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus (RBIV), and immune stimulants (LPS and poly (I:C)). Significant changes in RbSTAT3 transcription were also observed in response to tissue injury. In addition, the transcriptional up-regulation of RbSTAT3 was detected in rock bream

  11. Inhibition of FoxO transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy

    PubMed Central

    Reed, Sarah A.; Sandesara, Pooja B.; Senf, Sarah M.; Judge, Andrew R.

    2012-01-01

    Cachexia is characterized by inexorable muscle wasting that significantly affects patient prognosis and increases mortality. Therefore, understanding the molecular basis of this muscle wasting is of significant importance. Recent work showed that components of the forkhead box O (FoxO) pathway are increased in skeletal muscle during cachexia. In the current study, we tested the physiological significance of FoxO activation in the progression of muscle atrophy associated with cachexia. FoxO-DNA binding dependent transcription was blocked in the muscles of mice through injection of a dominant negative (DN) FoxO expression plasmid prior to inoculation with Lewis lung carcinoma cells or the induction of sepsis. Expression of DN FoxO inhibited the increased mRNA levels of atrogin-1, MuRF1, cathepsin L, and/or Bnip3 and inhibited muscle fiber atrophy during cancer cachexia and sepsis. Interestingly, during control conditions, expression of DN FoxO decreased myostatin expression, increased MyoD expression and satellite cell proliferation, and induced fiber hypertrophy, which required de novo protein synthesis. Collectively, these data show that FoxO-DNA binding-dependent transcription is necessary for normal muscle fiber atrophy during cancer cachexia and sepsis, and further suggest that basal levels of FoxO play an important role during normal conditions to depress satellite cell activation and limit muscle growth.—Reed, S. A., Sandesara, P. B., Senf, S. F., Judge, A. R. Inhibition of FoxO transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy. PMID:22102632

  12. In vivo simultaneous transcriptional activation of multiple genes in the brain using CRISPR-dCas9-activator transgenic mice.

    PubMed

    Zhou, Haibo; Liu, Junlai; Zhou, Changyang; Gao, Ni; Rao, Zhiping; Li, He; Hu, Xinde; Li, Changlin; Yao, Xuan; Shen, Xiaowen; Sun, Yidi; Wei, Yu; Liu, Fei; Ying, Wenqin; Zhang, Junming; Tang, Cheng; Zhang, Xu; Xu, Huatai; Shi, Linyu; Cheng, Leping; Huang, Pengyu; Yang, Hui

    2018-03-01

    Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.

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

    PubMed

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    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(+)-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.

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

    SciTech Connect

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi

    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.

  15. Efficient activation of transcription in yeast by the BPV1 E2 protein.

    PubMed Central

    Stanway, C A; Sowden, M P; Wilson, L E; Kingsman, A J; Kingsman, S M

    1989-01-01

    The full-length gene product encoded by the E2 open reading frame (ORF) of bovine papillomavirus type 1 (BPV1) is a transcriptional transactivator. It is believed to mediate its effect on the BPV1 long control region (LCR) by binding to motifs with the consensus sequence ACCN6GGT. The minimal functional cis active site, called the E2 response element (E2RE), in mammalian cells comprises two copies of this motif. Here we have shown that E2 can function in Saccharomyces cerevisiae by placing an E2RE upstream of a synthetic yeast assay promoter which consists of a TATA motif and an mRNA initiation site, spaced correctly. This E2RE-minimal promoter is only transcriptionally active in the presence of E2 protein and the resulting mRNA is initiated at the authentic start site. This is the first report of a mammalian viral transactivator functioning in yeast. The level of activation by E2 via the E2RE was the same as observed with the highly efficient authentic PGK promoter where the upstream activation sequence is composed of three distinct elements. Furthermore a single E2 motif which is insufficient in mammalian cells as an activation site was as efficiently utilized in yeast as the E2RE (2 motifs). Previous studies have shown that mammalian cellular activators can function in yeast and our data now extend this to viral-specific activators. Our data indicate however that while the mechanism of transactivation is broadly conserved there may be significant differences at the detailed level. Images PMID:2539584

  16. Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13.

    PubMed

    Choi, Jeong June; Park, Bo-Kyung; Park, Sunyoung; Yun, Chi-Young; Kim, Dong Hee; Kim, Jin Sook; Hwang, Eun Sook; Jin, Mirim

    2009-03-01

    Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a wellknown antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

  17. Quantitative evaluation of hepatic cytochrome P4501A transcript, protein, and catalytic activity in the striped sea bream (Lithognathus mormyrus).

    PubMed

    Tom, Moshe; Shmul, Merav; Shefer, Edna; Chen, Nir; Slor, Hanoch; Rinkevich, Baruch; Herut, Barak

    2003-09-01

    Hepatic cytochrome P4501A (CYP1A) expression was partially characterized in the striped sea bream (Lithognathus mormyrus) from the Mediterranean coast of Israel as part of the process of establishing the CYP1A gene as an environmental biomarker. Reverse transcription-competitive polymerase chain reaction, competitive enzyme-linked immunosorbent assay, and ethoxyresorufin O-deethylase (EROD) assay were used for evaluating transcript, protein, and catalytic activity levels, respectively, in absolute units. Highest elucidated transcript, protein, and catalytic activity levels were 0.264 +/- SD 0.084 fmol/microg total RNA, 0.88 +/- 0.52 pmol/microg total protein, and 1.11 +/- 0.52 pmol resorufin/min/microg total protein, respectively, and the lower levels were 0.009 +/- 0.007 fmol/microg total RNA, 0.17 +/- 0.08 pmol/microg total protein, and 0.11 +/- 0.06 pmol resorufin/min/microg total protein, respectively, demonstrating substantial induction potential. All alternate pairs of seven examined field samples, revealing a transcript-level ratio higher than 1.7, also demonstrated a significant difference between their transcript levels, indicating a potential to detect relatively small biomarker changes (1.7-fold) caused by environmental effects. Simultaneous triple measurements of transcript, protein, and catalytic activity were carried out in individuals from two field samples and during a 318-d decay experiment. Fish from the field samples revealed significant alternate bivariate correlation between transcript, protein, and enzymatic activity. Conflicting results were found when analyzing the decay experiment, in which both protein and catalytic activity levels decreased significantly to basal levels, in contrast to no significant change in transcript levels throughout the experiment. No significant difference was observed between males and females regarding the levels of CYP1A transcript, protein, and EROD.

  18. The transcriptional activator ZNF143 is essential for normal development in zebrafish

    PubMed Central

    2012-01-01

    Background ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. Results The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. Conclusions Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development. PMID:22268977

  19. The transcriptional activator ZNF143 is essential for normal development in zebrafish.

    PubMed

    Halbig, Kari M; Lekven, Arne C; Kunkel, Gary R

    2012-01-23

    ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development.

  20. Signal transducers and activators of transcription (STATs): Novel targets of chemopreventive and chemotherapeutic drugs.

    PubMed

    Klampfer, Lidija

    2006-03-01

    A family of latent cytoplasmic transcription factors, signal transducers and activators of transcription (STATs), mediates the responsiveness of cells to several cytokines and growth factors. Although mutations of STATs have not been described in human tumors, the activity of several members of the family, such as STAT1, STAT3 and STAT5, is deregulated in a variety of human tumors. STAT3 and STAT5 acquire oncogenic potential through constitutive phosphorylation on tyrosine, and their activity has been shown to be required to sustain a transformed phenotype. Disruption of STAT3 and STAT5 signaling in transformed cells therefore represents an excellent opportunity for targeted cancer therapy. In contrast to STAT3 and STAT5, STAT1 negatively regulates cell proliferation and angiogenesis and thereby inhibits tumor formation. Consistent with its tumor suppressive properties, STAT1 and its downstream targets have been shown to be reduced in a variety of human tumors and STAT1 deficient mice are highly susceptible to tumor formation. In recent years we have gained mechanistic understanding of the pathways whereby STATs convey signals from the cytoplasm to the nucleus. In addition, several endogenous regulators of the JAK/STAT pathway have been described - and their mechanism of action revealed - that profoundly affect signaling by STATs. Both should greatly facilitate the design of drugs with potential to modulate STAT signaling and to restore the homeostasis in tissues where STATs have gone awry.

  1. Capsaicin represses transcriptional activity of β-catenin in human colorectal cancer cells

    PubMed Central

    Lee, Seong-Ho; Richardson, Raphael L.; Dashwood, Roderick H.; Baek, Seung Joon

    2011-01-01

    Capsaicin is a pungent ingredient in chili red peppers and has been linked to suppression of growth in various cancer cells. However, the underlying mechanism(s) by which capsaicin induces growth arrest and apoptosis of cancer cells is not completely understood. In the present study, we investigated whether capsaicin alters β-catenin-dependent signaling in human colorectal cancer cells in vitro. Exposure of SW480, LoVo, and HCT-116 cells to capsaicin suppressed cell proliferation. Transient transfection with a β-catenin/T-cell factor (TCF)-responsive reporter indicated that capsaicin suppressed the transcriptional activity of β-catenin/TCF. Capsaicin treatment resulted in a decrease of intracellular β-catenin levels and a reduction of transcripts from the β-catenin gene (CTNNB1). These results were confirmed by a reduced luciferase reporter activity driven by promoter-reporter construct containing the promoter region of the Catnb gene. In addition, capsaicin destabilized β-catenin through enhancement of proteosomal-dependent degradation. Western blot and immunoprecipitation studies indicated that capsaicin treatment suppressed TCF-4 expression and disrupted the interaction of TCF-4 and β-catenin. This study identifies a role for the β-catenin/TCF-dependent pathway that potentially contributes to the anti-cancer activity of capsaicin in human colorectal cancer cells. PMID:21764279

  2. Retrotransposon Tf1 is targeted to pol II promoters by transcription activators

    PubMed Central

    Leem, Young-Eun; Ripmaster, Tracy; Kelly, Felice; Ebina, Hirotaka; Heincelman, Marc; Zhang, Ke; Grewal, Shiv I. S.; Hoffman, Charles S.; Levin, Henry L.

    2008-01-01

    SUMMARY The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration. We found that Tf1 was targeted specifically to the promoters of pol II transcribed genes. A detailed analysis of integration in plasmids that contained either ade6 or fbp1 revealed insertions occurred in the promoters at positions where transcription factors bound. Further experiments revealed that the activator Atf1p and its binding site were required for directing integration to the promoter of fbp1. An interaction between Tf1 integrase and Atf1p was observed indicating that integration at fbp1 was mediated by the activator bound to its promoter. Surprisingly we found Tf1 contained sequences that activated transcription and these substituted for elements of the ade6 promoter disrupted by integration. PMID:18406330

  3. Retrotransposon Tf1 is targeted to Pol II promoters by transcription activators.

    PubMed

    Leem, Young-Eun; Ripmaster, Tracy L; Kelly, Felice D; Ebina, Hirotaka; Heincelman, Marc E; Zhang, Ke; Grewal, Shiv I S; Hoffman, Charles S; Levin, Henry L

    2008-04-11

    The LTR-retrotransposon Tf1 preserves the coding capacity of its host Schizosaccharomyces pombe by integrating upstream of open reading frames (ORFs). To determine which features of the target sites were recognized by the transposon, we introduced plasmids containing candidate insertion sites into S. pombe and mapped the positions of integration. We found that Tf1 was targeted specifically to the promoters of Pol II-transcribed genes. A detailed analysis of integration in plasmids that contained either ade6 or fbp1 revealed insertions occurred in the promoters at positions where transcription factors bound. Further experiments revealed that the activator Atf1p and its binding site were required for directing integration to the promoter of fbp1. An interaction between Tf1 integrase and Atf1p was observed, indicating that integration at fbp1 was mediated by the activator bound to its promoter. Surprisingly, we found Tf1 contained sequences that activated transcription, and these substituted for elements of the ade6 promoter disrupted by integration.

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

    SciTech Connect

    Zhang, Yingyi; Zhao, Yu; Li, Leilei

    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)more » 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.« less

  5. Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors.

    PubMed Central

    Burk, O; Mink, S; Ringwald, M; Klempnauer, K H

    1993-01-01

    The retroviral oncogene v-myb encodes a transcriptional activator which is responsible for the activation of the mim-1 gene in myelomonocytic cells transformed by v-myb. The mim-1 promoter contains several myb consensus binding sites and has previously been shown to be regulated directly by v-myb. Here we report that the mim-1 gene is activated synergistically by v-myb and different C/EBP transcription factors. We have cloned a chicken C/EBP-related gene that is highly expressed in myeloid cells and identified it as the chicken homolog of C/EBP beta. A dominant-negative variant of chicken C/EBP beta interferes with the v-myb induced activation of the mim-1 gene in these cells, suggesting that C/EBP beta or another C/EBP transcription factor is required for the activation of mim-1 by v-myb. We found that C/EBP beta and other C/EBP transcription factors confer to fibroblasts the ability to induce the mim-1 gene in the presence of v-myb. Finally we show that, in contrast to v-myb, c-myb synergizes with C/EBP transcription factors only at low concentrations of c-myb protein. Our results suggest a role for C/EBP beta, and possibly for other C/EBP transcription factors, in v-myb function and in myeloid-specific gene activation. Images PMID:8491193

  6. Molecular Genetic Analysis of Activation-tagged Transcription Factors Thought to be Involved in Photomorphogenesis

    SciTech Connect

    Neff, Michael

    2011-06-23

    Plants utilize light as a source of information via families of photoreceptors such as the red/far-red absorbing phytochromes (PHY) and the blue/UVA absorbing cryptochromes (CRY). The main goal of the Neff lab is to use molecular-genetic mutant screens to elucidate signaling components downstream of these photoreceptors. Activation-tagging mutagenesis led to the identification of two putative transcription factors that may be involved in both photomorphogenesis and hormone signaling pathways. sob1-D (suppressor of phyB-dominant) mutant phenotypes are caused by the over-expression of a Dof transcription factor previously named OBP3. Our previous studies indicate that OBP3 is a negative regulator of light-mediated cotyledonmore » expansion and may be involved in modulating responsiveness to the growth-regulating hormone auxin. The sob2-D mutant uncovers a role for LEP, a putative AP2/EREBP-like transcription factor, in seed germination, hypocotyl elongation and responsiveness to the hormone abscisic acid. Based on photobiological and genetic analysis of OBP3-knockdown and LEP-null mutations, we hypothesize that these transcription factors are involved in both light-mediated seedling development and hormone signaling. To examine the role that these genes play in photomorphogenesis we will: 1) Further explore the genetic role of OBP3 in cotyledon/leaf expansion and other photomorphogenic processes as well as examine potential physical interactions between OBP3 and CRY1 or other signaling components that genetically interact with this transcription factor 2) Test the hypothesis that OBP3 is genetically involved in auxin signaling and root development as well as examine the affects of this hormone and light on OBP3 protein accumulation. 3) Test the hypothesis that LEP is involved in seed germination, seedling photomorphogenesis and hormone signaling. Together these experiments will lead to a greater understanding of the complexity of interactions between photoreceptors

  7. Characterization of a new ARID family transcription factor (Brightlike/ARID3C) that co-activates Bright/ARID3A-mediated immunoglobulin gene transcription

    PubMed Central

    Tidwell, Josephine A.; Schmidt, Christian; Heaton, Phillip; Wilson, Van; Tucker, Philip W.

    2011-01-01

    Two members, Bright/ARID3A and Bdp/ARID3B, of the ARID (AT-Rich Interaction Domain) transcription family are distinguished by their ability to specifically bind to DNA and to self-associate via a second domain, REKLES. Bright and Bdp positively regulate immunoglobulin heavy chain gene (IgH) transcription by binding to AT-rich motifs within Matrix Associating Regions (MARs) residing within a subset of VH promoters and the Eµ intronic enhancer. In addition, REKLES provides Bright nuclear export function, and a small pool of Bright is directed to plasma membrane sub-domains/lipid rafts where it associates with and modulates signaling of the B cell antigen receptor (BCR). Here, we characterize a third, highly conserved, physically condensed ARID3 locus, Brightlike/ARID3C. Brightlike encodes two alternatively spliced, SUMO-I-modified isoforms that include or exclude (Δ6) the REKLES-encoding exon 6. Brightlike transcripts and proteins are expressed preferentially within B lineage lymphocytes and coordinate with highest Bright expression--in activated follicular B cells. Brightlike, but not BrightlikeΔ6, undergoes nuclear-cytoplasmic shuttling with a fraction localizing within lipid rafts following BCR stimulation. Brightlike, but not BrightlikeΔ6, associates with Bright in solution, at common DNA binding sites in vitro, and is enriched at Bright binding sites in chromatin. Although possessing little transactivation capacity of its own, Brightlike significantly co-activates Bright-dependent IgH transcription with maximal activity mediated by the unsumoylated form. In sum, this report introduces Brightlike as an additional functional member of the family of ARID proteins, which should be considered in regulatory circuits, previously ascribed to be mediated by Bright. PMID:21955986

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

    PubMed Central

    2012-01-01

    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. PMID:23046558

  9. Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

    PubMed

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

    2017-08-30

    Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the

  10. Structure-Function Analysis of the Drosophila melanogaster Caudal Transcription Factor Provides Insights into Core Promoter-preferential Activation.

    PubMed

    Shir-Shapira, Hila; Sharabany, Julia; Filderman, Matan; Ideses, Diana; Ovadia-Shochat, Avital; Mannervik, Mattias; Juven-Gershon, Tamar

    2015-07-10

    Regulation of RNA polymerase II transcription is critical for the proper development, differentiation, and growth of an organism. The RNA polymerase II core promoter is the ultimate target of a multitude of transcription factors that control transcription initiation. Core promoters encompass the RNA start site and consist of functional elements such as the TATA box, initiator, and downstream core promoter element (DPE), which confer specific properties to the core promoter. We have previously discovered that Drosophila Caudal, which is a master regulator of genes involved in development and differentiation, is a DPE-specific transcriptional activator. Here, we show that the mouse Caudal-related homeobox (Cdx) proteins (mCdx1, mCdx2, and mCdx4) are also preferential core promoter transcriptional activators. To elucidate the mechanism that enables Caudal to preferentially activate DPE transcription, we performed structure-function analysis. Using a systematic series of deletion mutants (all containing the intact DNA-binding homeodomain) we discovered that the C-terminal region of Caudal contributes to the preferential activation of the fushi tarazu (ftz) Caudal target gene. Furthermore, the region containing both the homeodomain and the C terminus of Caudal was sufficient to confer core promoter-preferential activation to the heterologous GAL4 DNA-binding domain. Importantly, we discovered that Drosophila CREB-binding protein (dCBP) is a co-activator for Caudal-regulated activation of ftz. Strikingly, dCBP conferred the ability to preferentially activate the DPE-dependent ftz reporter to mini-Caudal proteins that were unable to preferentially activate ftz transcription themselves. Taken together, it is the unique combination of dCBP and Caudal that enables the co-activation of ftz in a core promoter-preferential manner. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator toxT in response to culture conditions.

    PubMed

    Medrano, A I; DiRita, V J; Castillo, G; Sanchez, J

    1999-05-01

    Vibrio cholerae El Tor require special in vitro culture conditions, consisting of an initial static growth period followed by shift to shaking (AKI conditions), for expression of cholera toxin (CT) and toxin coregulated pili (TCP). ToxT, a regulator whose initial transcription depends on the ToxR regulator, positively modulates expression of CT and TCP. To help understand control of CT and TCP in El Tor vibrios, we monitored ctxAB and ToxR-dependent toxT transcription by time course primer extension assays. AKI conditions stimulated CT synthesis with an absence of ctxAB transcription during static growth followed by induction upon shaking. ToxR-dependent toxT transcription was induced at the end of the static growth period but was transient, stopping shortly after shaking was initiated but, interestingly, also if the static phase was prolonged. Immunoblot assays showed that ToxR protein levels were not coincidentally transient, implying a protein on/off switch mechanism for ToxR. Despite the transient activation by ToxR, transcription of ctxAB was maintained during shaking. This finding suggested continued toxT expression, possibly through relay transcription from another promoter. The 12.6-kb distant upstream tcpA promoter responsible for expression of the TCP operon has been proposed to provide an alternate toxT message by readthrough transcription. Activation of the tcpA promoter is supported by increased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription of toxT from tcpA would be compatible with reverse transcription-PCR evidence for a toxT mRNA at times when ToxR-dependent transcription was no longer detectable by primer extension.

  12. Genetic variation in the MITF promoter affects skin colour and transcriptional activity in black-boned chickens.

    PubMed

    Wang, G; Liao, J; Tang, M; Yu, S

    2018-02-01

    1. Microphthalmia-associated transcription factor (MITF) plays a pivotal role in melanocyte development by regulating the transcription of major pigmentation enzymes (e.g. TYR, TYRP1 and DCT). A single-nucleotide polymorphism (SNP), c.-638T>C, was identified in the MITF promoter, and genotyping of a population (n = 426) revealed that SNP c.-638T>C was associated with skin colour in black-boned chickens. 2. Individuals with genotypes CC and TC exhibited greater MTIF expression than those with genotype TT. Luciferase assays also revealed that genotype CC and TC promoters had higher activity levels than genotype TT. Expression of melanogenesis-related gene (TYR) was higher in the skin of chickens with the CC and CT genotype compared to TT chickens (P < 0.05). 3. Transcription factor-binding site analyses showed that the c.-638C allele contains a putative binding site for transcription factor sterol regulatory element-binding transcription factor 2, aryl hydrocarbon receptor nuclear translocator, transcription factor binding to IGHM enhancer 3 and upstream transcription factor 2. In contrast, the c.-638T allele contains binding sites for Sp3 transcription factor and Krüppel-like factor 1. 4. It was concluded that MITF promoter polymorphisms affected chicken skin colour. SNP c.-638T>C could be used for the marker-assisted selection of skin colour in black-boned chicken breeding.

  13. The Ability to Associate with Activation Domains in vitro is not Required for the TATA Box-Binding Protein to Support Activated Transcription in vivo

    NASA Astrophysics Data System (ADS)

    Tansey, William P.; Herr, Winship

    1995-11-01

    The TATA box-binding protein (TBP) interacts in vitro with the activation domains of many viral and cellular transcription factors and has been proposed to be a direct target for transcriptional activators. We have examined the functional relevance of activator-TBP association in vitro to transcriptional activation in vivo. We show that alanine substitution mutations in a single loop of TBP can disrupt its association in vitro with the activation domains of the herpes simplex virus activator VP16 and of the human tumor suppressor protein p53; these mutations do not, however, disrupt the transcriptional response of TBP to either activation domain in vivo. Moreover, we show that a region of VP16 distinct from its activation domain can also tightly associate with TBP in vitro, but fails to activate transcription in vivo. These data suggest that the ability of TBP to interact with activation domains in vitro is not directly relevant to its ability to support activated transcription in vivo.

  14. Shikimate Induced Transcriptional Activation of Protocatechuate Biosynthesis Genes by QuiR, a LysR-Type Transcriptional Regulator, in Listeria monocytogenes.

    PubMed

    Prezioso, Stephanie M; Xue, Kevin; Leung, Nelly; Gray-Owen, Scott D; Christendat, Dinesh

    2018-04-27

    Listeria monocytogenes is a common foodborne bacterial pathogen that contaminates plant and animal consumable products. The persistent nature of L. monocytogenes is associated with millions of dollars in food recalls annually. Here, we describe the role of shikimate in directly modulating the expression of genes encoding enzymes for the conversion of quinate and shikimate metabolites to protocatechuate. In L. monocytogenes, these genes are found within two operons, named qui1 and qui2. In addition, a gene named quiR, encoding a LysR-Type Transcriptional Regulator (QuiR), is located immediately upstream of the qui1 operon. Transcriptional lacZ-promoter fusion experiments show that QuiR induces gene expression of both qui1 and qui2 operons in the presence of shikimate. Furthermore, co-crystallization of the QuiR effector binding domain in complex with shikimate provides insights into the mechanism of activation of this regulator. Together these data show that upon shikimate accumulation, QuiR activates the transcription of genes encoding enzymes involved in shikimate and quinate utilization for the production of protocatechuate. Furthermore, the accumulation of protocatechuate leads to the inhibition of Listeria growth. Since protocatechuate is not known to be utilized by Listeria, its role is distinct from those established in other bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Tanshinone IIA inhibits cervix carcinoma stem cells migration and invasion via inhibiting YAP transcriptional activity.

    PubMed

    Qin, Jinghao; Shi, Hongbing; Xu, Yanjie; Zhao, Fang; Wang, Qing

    2018-06-14

    This study aims to explore the effects and related mechanisms of Tanshinone IIA in cervix carcinoma (CC) stemness-like cells migration, invasion, stemness and chemotherapeutical sensitivity. Here, we found that Tanshinone IIA suppressed CC stemness-like cells migration and invasion in a concentration- and time-dependent manner. And consistent results were obtained in CC cells stemness characterized as the decrease of CC stemness markers expression and cells spheroid formation ability. Mechanistically, we found that Tanshinone IIA suppressed RNA binding protein HuR translocation from nuclear to cytoplasm, and thus reduced YAP mRNAs stability and transcriptional activity. Importantly, overexpression YAP-5SA rescued the inhibition of Tanshinone IIA on CC cells stemness. Furthermore, Tanshinone IIA enhanced adriamycin sensitivity in CC stemness-like cells, this effect was attenuated by YAP-5SA overexpression too. Therefore, Tanshinone IIA could suppress CC stemness-like cells migration and invasion by inhibiting YAP transcriptional activity. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  16. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

    PubMed

    Joung, Julia; Konermann, Silvana; Gootenberg, Jonathan S; Abudayyeh, Omar O; Platt, Randall J; Brigham, Mark D; Sanjana, Neville E; Zhang, Feng

    2017-04-01

    Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.

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

    PubMed

    Franken, P; Lopez-Molina, L; Marcacci, L; Schibler, U; Tafti, M

    2000-01-15

    Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues. Mice lacking DBP display a shorter circadian period in locomotor activity and are less active. Thus, although DBP is not essential for circadian rhythm generation, it does modulate important clock outputs. We studied the role of DBP in the circadian and homeostatic aspects of sleep regulation by comparing DBP deficient mice (dbp-/-) with their isogenic controls (dbp+/+) under light-dark (LD) and constant-dark (DD) baseline conditions, as well as after sleep loss. Whereas total sleep duration was similar in both genotypes, the amplitude of the circadian modulation of sleep time, as well as the consolidation of sleep episodes, was reduced in dbp-/- under both LD and DD conditions. Quantitative EEG analysis demonstrated a marked reduction in the amplitude of the sleep-wake-dependent changes in slow-wave sleep delta power and an increase in hippocampal theta peak frequency in dbp-/- mice. The sleep deprivation-induced compensatory rebound of EEG delta power was similar in both genotypes. In contrast, the rebound in paradoxical sleep was significant in dbp+/+ mice only. It is concluded that the transcriptional regulatory protein DBP modulates circadian and homeostatic aspects of sleep regulation.

  18. Single-molecule quantification of lipotoxic expression of activating transcription factor 3

    PubMed Central

    Wilson, Dennis W.; Rutledge, John C.

    2014-01-01

    Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500/h. After three hours we detected 33,090 ± 3,491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at ~3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipolysis products occurs in large aggregates. PMID:25189785

  19. Chronic ethanol consumption inhibits glucokinase transcriptional activity by Atf3 and triggers metabolic syndrome in vivo.

    PubMed

    Kim, Ji Yeon; Hwang, Joo-Yeon; Lee, Dae Yeon; Song, Eun Hyun; Park, Keon Jae; Kim, Gyu Hee; Jeong, Eun Ae; Lee, Yoo Jeong; Go, Min Jin; Kim, Dae Jin; Lee, Seong Su; Kim, Bong-Jo; Song, Jihyun; Roh, Gu Seob; Gao, Bin; Kim, Won-Ho

    2014-09-26

    Chronic ethanol consumption induces pancreatic β-cell dysfunction through glucokinase (Gck) nitration and down-regulation, leading to impaired glucose tolerance and insulin resistance, but the underlying mechanism remains largely unknown. Here, we demonstrate that Gck gene expression and promoter activity in pancreatic β-cells were suppressed by chronic ethanol exposure in vivo and in vitro, whereas expression of activating transcription factor 3 (Atf3) and its binding to the putative Atf/Creb site (from -287 to -158 bp) on the Gck promoter were up-regulated. Furthermore, in vitro ethanol-induced Atf3 inhibited the positive effect of Pdx-1 on Gck transcriptional regulation, enhanced recruitment of Hdac1/2 and histone H3 deacetylation, and subsequently augmented the interaction of Hdac1/Pdx-1 on the Gck promoter, which were diminished by Atf3 siRNA. In vivo Atf3-silencing reversed ethanol-mediated Gck down-regulation and β-cell dysfunction, followed by the amelioration of impaired glucose tolerance and insulin resistance. Together, we identified that ethanol-induced Atf3 fosters β-cell dysfunction via Gck down-regulation and that its loss ameliorates metabolic syndrome and could be a potential therapeutic target in treating type 2 diabetes. The Atf3 gene is associated with the induction of type 2 diabetes and alcohol consumption-induced metabolic impairment and thus may be the major negative regulator for glucose homeostasis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Genome-scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening

    PubMed Central

    Joung, Julia; Konermann, Silvana; Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Platt, Randall J.; Brigham, Mark D.; Sanjana, Neville E.; Zhang, Feng

    2017-01-01

    Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9–15 weeks followed by 4–5 weeks of validation. PMID:28333914

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

    PubMed

    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. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  2. Transcriptional activity of detoxification genes is altered by ultraviolet filters in Chironomus riparius.

    PubMed

    Martínez-Guitarte, José-Luis

    2018-03-01

    Ultraviolet (UV) filters are compounds used to prevent the damage produced by UV radiation in personal care products, plastics, etc. They have been associated with endocrine disruption, showing anti-estrogen activity in vertebrates and altering the ecdysone pathway in invertebrates. Although they have attracted the attention of multiple research teams there is a lack of data about how animals activate detoxification systems, especially in invertebrates. Here, analysis of the effects of two UV filters, benzophenone-3 (BP3) and 4-methylbenzylidene camphor (4MBC), on the transcriptional activity of nine genes covering the three steps of the detoxification process has been performed. Four cytochrome P450 genes belonging to different members of this family, five GST genes, and the multidrug resistance protein 1 (MRP1) gene were studied by RT-PCR to analyze their transcriptional activity in fourth instar larvae exposed to the UV filters for 8 and 24h. The obtained results show a differential response with downregulation of the different Cyp450s tested by 4MBC while BP3 seems not to modify their expression. On the other hand, some of the GST genes were affected by one or other of the filters, showing a less homogenous response. Finally, MRP1 was activated by both filters but at different times. These results demonstrate for first time that UV filters alter the expression of genes involved in the different steps of the detoxification process and that they can be processed by phase I enzymes other than Cyp450s. They also suggest that UV filters affect biotransformation processes, compromising the ability of the individual to respond to chemical stress, so further research is needed to know the extent of the damage that they can produce in the resistance of the cell to chemicals. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers

    SciTech Connect

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O., E-mail: pustylnyak@ngs.ru

    Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p′-DDT (85%) and o,p′-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CARmore » and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45β, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45β. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the β-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers. - Highlights: • DDT activated both CAR and ERα and their

  4. Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

    PubMed

    Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

    2016-08-01

    The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes

    PubMed Central

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-01-01

    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. PMID:25046113