Functional synthetic Antennapedia genes and the dual roles of YPWM motif and linker size in transcriptional activation and repression

PubMed Central

Papadopoulos, Dimitrios K.; Reséndez-Pérez, Diana; Cárdenas-Chávez, Diana L.; Villanueva-Segura, Karina; Canales-del-Castillo, Ricardo; Felix, Daniel A.; Fünfschilling, Raphael; Gehring, Walter J.

2011-01-01

Segmental identity along the anteroposterior axis of bilateral animals is specified by Hox genes. These genes encode transcription factors, harboring the conserved homeodomain and, generally, a YPWM motif, which binds Hox cofactors and increases Hox transcriptional specificity in vivo. Here we derive synthetic Drosophila Antennapedia genes, consisting only of the YPWM motif and homeodomain, and investigate their functional role throughout development. Synthetic peptides and full-length Antennapedia proteins cause head-to-thorax transformations in the embryo, as well as antenna-to-tarsus and eye-to-wing transformations in the adult, thus converting the entire head to a mesothorax. This conversion is achieved by repression of genes required for head and antennal development and ectopic activation of genes promoting thoracic and tarsal fates, respectively. Synthetic Antennapedia peptides bind DNA specifically and interact with Extradenticle and Bric-à-brac interacting protein 2 cofactors in vitro and ex vivo. Substitution of the YPWM motif by alanines abolishes Antennapedia homeotic function, whereas substitution of YPWM by the WRPW repressor motif, which binds the transcriptional corepressor Groucho, allows all proteins to act as repressors only. Finally, naturally occurring variations in the size of the linker between the homeodomain and YPWM motif enhance Antennapedia repressive or activating efficiency, emphasizing the importance of linker size, rather than sequence, for specificity. Our results clearly show that synthetic Antennapedia genes are functional in vivo and therefore provide powerful tools for synthetic biology. Moreover, the YPWM motif is necessary—whereas the entire N terminus of the protein is dispensable—for Antennapedia homeotic function, indicating its dual role in transcriptional activation and repression by recruiting either coactivators or corepressors. PMID:21712439

Mitochondrial functions mediate cellulase gene expression in Trichoderma reesei.

PubMed

Abrahão-Neto, J; Rossini, C H; el-Gogary, S; Henrique-Silva, F; Crivellaro, O; el-Dorry, H

1995-08-22

We examined the effects of inhibition of mitochondrial functions on the expression of two nuclear genes encoding the extracellular cellobiohydrolase I (cbh1) and endoglucanase I (egl1) of the cellulase system of the filamentous fungus Trichoderma reesei. The cbh1 and egl1 transcripts are repressed at a low oxygen tension, and by glucose at a concentration known to repress mitochondrial respiration. The transcripts are also down-regulated by chemical agents known to dissipate the proton electrochemical gradient of the inner mitochondrial membrane and blocking of the electron-transport chain, such as DNP and KCN, respectively. These results suggest that expression of those transcripts is influenced by the physiological state of the mitochondria. In addition, heterologous gene fusion shows that the sensitivity of the expression of those transcripts to the functional state of the mitochondria is transcriptionally controlled through the 5'-flanking DNA sequence of those genes.

The transcription factor DREAM represses A20 and mediates inflammation

PubMed Central

Tiruppathi, Chinnaswamy; Soni, Dheeraj; Wang, Dong-Mei; Xue, Jiaping; Singh, Vandana; Thippegowda, Prabhakar B.; Cheppudira, Bopaiah P.; Mishra, Rakesh K.; DebRoy, Auditi; Qian, Zhijian; Bachmaier, Kurt; Zhao, Youyang; Christman, John W.; Vogel, Stephen M.; Ma, Averil; Malik, Asrar B.

2014-01-01

Here we show that the transcription-repressor DREAM binds to the A20 promoter to repress the expression of A20, the deubiquitinase suppressing inflammatory NF-κB signaling. DREAM-deficient (Dream−/−) mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, USF1 binding to the DRE-associated E-box domain activated A20 expression in response to inflammatory stimuli. These studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy in diseases such as acute lung injury associated with unconstrained NF-κB activity. PMID:24487321

Rice NAD+-dependent histone deacetylase OsSRT1 represses glycolysis and regulates the moonlighting function of GAPDH as a transcriptional activator of glycolytic genes.

PubMed

Zhang, Hua; Zhao, Yu; Zhou, Dao-Xiu

2017-12-01

Sirtuins, a family of proteins with homology to the yeast silent information regulator 2 (Sir2), are NAD+-dependent histone deacetylases and play crucial roles in energy sensing and regulation in yeast and animal cells. Plants are autotrophic organisms and display distinct features of carbon and energy metabolism. It remains largely unexplored whether and how plant cells sense energy/redox status to control carbon metabolic flux under various growth conditions. In this work, we show that the rice nuclear sirtuin OsSRT1 not only functions as an epigenetic regulator to repress glycolytic genes expression and glycolysis in seedlings, but also inhibits transcriptional activity of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) that is enriched on glycolytic genes promoters and stimulates their expression. We show that OsSRT1 reduces GAPDH lysine acetylation and nuclear accumulation that are enhanced by oxidative stress. Mass spectrometry identified six acetylated lysines regulated by OsSRT1. OsSRT1-dependent lysine deacetylation of OsGAPDH1 represses transcriptional activity of the protein. The results indicate that OsSRT1 represses glycolysis by both regulating epigenetic modification of histone and inhibiting the moonlighting function of GAPDH as a transcriptional activator of glycolytic genes in rice. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

PubMed Central

Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

PubMed

Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Function of multiple Lis-Homology domain/WD-40 repeat-containing proteins in feed-forward transcriptional repression by silencing mediator for retinoic and thyroid receptor/nuclear receptor corepressor complexes.

PubMed

Choi, Hyo-Kyoung; Choi, Kyung-Chul; Kang, Hee-Bum; Kim, Han-Cheon; Lee, Yoo-Hyun; Haam, Seungjoo; Park, Hyoung-Gi; Yoon, Ho-Geun

2008-05-01

Lis-homology (LisH) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal LisH domain in transducin beta-like protein 1 and its receptor (TBL1 and TBLR1) led us to examine the role of this domain in transcriptional repression. Here we show that multiple beta-transducin (WD-40) repeat-containing proteins interact to form oligomers in solution and that oligomerization depends on the presence of the LisH domain in each protein. Repression of transcription, as assayed using Gal4 fusion proteins, also depended on the presence of the LisH domain, suggesting that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, we show that the LisH domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor complex. Mutations in conserved residues in the LisH motif of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the LisH motif in transcriptional repression. Our results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. Finally, we observed that the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. We also found the specific interaction UbcH/E2 with TBL1, but not RbAp46/48. Altogether, our results thus indicate that the presence of multiple LisH/WD-40 repeat containing proteins is exclusive to nuclear receptor corepressor/ silencing mediator for retinoic and thyroid receptor complexes compared with other class 1 histone deacetylase-containing corepessor complexes.

Histone methyltransferase Ash1L mediates activity-dependent repression of neurexin-1α

PubMed Central

Zhu, Τao; Liang, Chen; Li, Dongdong; Tian, Miaomiao; Liu, Sanxiong; Gao, Guanjun; Guan, Ji-Song

2016-01-01

Activity-dependent transcription is critical for the regulation of long-term synaptic plasticity and plastic rewiring in the brain. Here, we report that the transcription of neurexin1α (nrxn1α), a presynaptic adhesion molecule for synaptic formation, is regulated by transient neuronal activation. We showed that 10 minutes of firing at 50 Hz in neurons repressed the expression of nrxn1α for 24 hours in a primary cortical neuron culture through a transcriptional repression mechanism. By performing a screening assay using a synthetic zinc finger protein (ZFP) to pull down the proteins enriched near the nrxn1α promoter region in vivo, we identified that Ash1L, a histone methyltransferase, is enriched in the nrxn1α promoter. Neuronal activity triggered binding of Ash1L to the promoter and enriched the histone marker H3K36me2 at the nrxn1α promoter region. Knockout of Ash1L in mice completely abolished the activity-dependent repression of nrxn1α. Taken together, our results reveal that a novel process of activity-dependent transcriptional repression exists in neurons and that Ash1L mediates the long-term repression of nrxn1α, thus implicating an important role for epigenetic modification in brain functioning. PMID:27229316

Cutaneous Papillomavirus E6 oncoproteins associate with MAML1 to repress transactivation and NOTCH signaling

PubMed Central

Brimer, Nicole; Lyons, Charles; Wallberg, Annika E.; Vande Pol, Scott B.

2011-01-01

Papillomavirus E6 oncoproteins associate with LXXLL motifs on target cellular proteins to alter their function. Using a proteomic approach, we found the E6 oncoproteins of cutaneous papillomaviruses Bovine Papillomavirus Type 1 (BE6) and HPV types 1 and 8 (1E6 and 8E6) associated with the MAML1 transcriptional co-activator. All three E6 proteins bind to an acidic LXXLL motif at the carboxy-terminus of MAML1 and repress transactivation by MAML1. MAML1 is best known as the co-activator and effector of NOTCH induced transcription, and BPV-1 E6 represses synthetic NOTCH responsive promoters, endogenous NOTCH responsive promoters, and is found in a complex with MAML1 in stably transformed cells. BPV-1 induced papillomas show characteristics of repressed NOTCH signal transduction, including suprabasal expression of integrins, talin, and basal type keratins, and delayed expression of the NOTCH dependent HES1 transcription factor. These observations give rise to a model whereby papillomavirus oncoproteins including BPV-1 E6 and the cancer associated HPV-8 E6 repress Notch induced transcription, thereby delaying keratinocyte differentiation. PMID:22249263

Epigenetic silencing of Bim transcription by Spi-1/PU.1 promotes apoptosis resistance in leukaemia

PubMed Central

Ridinger-Saison, M; Evanno, E; Gallais, I; Rimmelé, P; Selimoglu-Buet, D; Sapharikas, E; Moreau-Gachelin, F; Guillouf, C

2013-01-01

Deregulation of transcriptional networks contributes to haematopoietic malignancies. The transcription factor Spi-1/PU.1 is a master regulator of haematopoiesis and its alteration leads to leukaemia. Spi-1 overexpression inhibits differentiation and promotes resistance to apoptosis in erythroleukaemia. Here, we show that Spi-1 inhibits mitochondrial apoptosis in vitro and in vivo through the transcriptional repression of Bim, a proapoptotic factor. BIM interacts with MCL-1 that behaves as a major player in the survival of the preleukaemic cells. The repression of BIM expression reduces the amount of BIM-MCL-1 complexes, thus increasing the fraction of potentially active antiapoptotic MCL-1. We then demonstrate that Spi-1 represses Bim transcription by binding to the Bim promoter and by promoting the trimethylation of histone 3 on lysine 27 (H3K27me3, a repressive histone mark) on the Bim promoter. The PRC2 repressive complex of Polycomb is directly responsible for the deposit of H3K27me3 mark at the Bim promoter. SUZ12 and the histone methyltransferase EZH2, two PRC2 subunits bind to the Bim promoter at the same location than H3K27me3, distinct of the Spi-1 DNA binding site. As Spi-1 interacts with SUZ12 and EZH2, these results indicate that Spi-1 modulates the activity of PRC2 without directly recruiting the complex to the site of its activity on the chromatin. Our results identify a new mechanism whereby Spi-1 represses transcription and provide mechanistic insights on the antiapoptotic function of a transcription factor mediated by the epigenetic control of gene expression. PMID:23852375

The transcription factor Mlc promotes Vibrio cholerae biofilm formation through repression of phosphotransferase system components.

PubMed

Pickering, Bradley S; Lopilato, Jane E; Smith, Daniel R; Watnick, Paula I

2014-07-01

The phosphoenol phosphotransferase system (PTS) is a multicomponent signal transduction cascade that regulates diverse aspects of bacterial cellular physiology in response to the availability of high-energy sugars in the environment. Many PTS components are repressed at the transcriptional level when the substrates they transport are not available. In Escherichia coli, the transcription factor Mlc (for makes large colonies) represses transcription of the genes encoding enzyme I (EI), histidine protein (HPr), and the glucose-specific enzyme IIBC (EIIBC(Glc)) in defined media that lack PTS substrates. When glucose is present, the unphosphorylated form of EIIBC(Glc) sequesters Mlc to the cell membrane, preventing its interaction with DNA. Very little is known about Vibrio cholerae Mlc. We found that V. cholerae Mlc activates biofilm formation in LB broth but not in defined medium supplemented with either pyruvate or glucose. Therefore, we questioned whether V. cholerae Mlc functions differently than E. coli Mlc. Here we have shown that, like E. coli Mlc, V. cholerae Mlc represses transcription of PTS components in both defined medium and LB broth and that E. coli Mlc is able to rescue the biofilm defect of a V. cholerae Δmlc mutant. Furthermore, we provide evidence that Mlc indirectly activates transcription of the vps genes by repressing expression of EI. Because activation of the vps genes by Mlc occurs under only a subset of the conditions in which repression of PTS components is observed, we conclude that additional inputs present in LB broth are required for activation of vps gene transcription by Mlc. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Distinct Residues Contribute to Motility Repression and Autoregulation in the Proteus mirabilis Fimbria-Associated Transcriptional Regulator AtfJ.

PubMed

Bode, Nadine J; Chan, Kun-Wei; Kong, Xiang-Peng; Pearson, Melanie M

2016-08-01

Proteus mirabilis contributes to a significant number of catheter-associated urinary tract infections, where coordinated regulation of adherence and motility is critical for ascending disease progression. Previously, the mannose-resistant Proteus-like (MR/P) fimbria-associated transcriptional regulator MrpJ has been shown to both repress motility and directly induce the transcription of its own operon; in addition, it affects the expression of a wide range of cellular processes. Interestingly, 14 additional mrpJ paralogs are included in the P. mirabilis genome. Looking at a selection of MrpJ paralogs, we discovered that these proteins, which consistently repress motility, also have nonidentical functions that include cross-regulation of fimbrial operons. A subset of paralogs, including AtfJ (encoded by the ambient temperature fimbrial operon), Fim8J, and MrpJ, are capable of autoinduction. We identified an element of the atf promoter extending from 487 to 655 nucleotides upstream of the transcriptional start site that is responsive to AtfJ, and we found that AtfJ directly binds this fragment. Mutational analysis of AtfJ revealed that its two identified functions, autoregulation and motility repression, are not invariably linked. Residues within the DNA-binding helix-turn-helix domain are required for motility repression but not necessarily autoregulation. Likewise, the C-terminal domain is dispensable for motility repression but is essential for autoregulation. Supported by a three-dimensional (3D) structural model, we hypothesize that the C-terminal domain confers unique regulatory capacities on the AtfJ family of regulators. Balancing adherence with motility is essential for uropathogens to successfully establish a foothold in their host. Proteus mirabilis uses a fimbria-associated transcriptional regulator to switch between these antagonistic processes by increasing fimbrial adherence while simultaneously downregulating flagella. The discovery of multiple related proteins, many of which also function as motility repressors, encoded in the P. mirabilis genome has raised considerable interest as to their functionality and potential redundancy in this organism. This study provides an important advance in this field by elucidating the nonidentical effects of these paralogs on a molecular level. Our mechanistic studies of one member of this group, AtfJ, shed light on how these differing functions may be conferred despite the limited sequence variety exhibited by the paralogous proteins. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interference of transcription across H-NS binding sites and repression by H-NS.

PubMed

Rangarajan, Aathmaja Anandhi; Schnetz, Karin

2018-05-01

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation. In vitro H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination, while in vivo inhibition of Rho resulted in a decrease of the genome occupancy by H-NS. Here we show that transcription directed across H-NS binding regions relieves H-NS (and H-NS/StpA) mediated repression of promoters in these regions. Further, we observed a correlation of transcription across the H-NS-bound region and de-repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS complex and/or dislodge H-NS from the DNA and thus relieve repression. Such an interference of transcription and H-NS mediated repression may imply that poorly transcribed AT-rich loci are prone to be repressed by H-NS, while efficiently transcribed loci escape repression. © 2018 John Wiley & Sons Ltd.

Influence of gene dosage and autoregulation of the regulatory genes INO2 and INO4 on inositol/choline-repressible gene transcription in the yeast Saccharomyces cerevisiae.

PubMed

Schwank, S; Hoffmann, B; Sch-uller, H J

1997-06-01

Expression of structural genes of phospholipid biosynthesis in yeast is mediated by the inositol/choline-responsive element (ICRE). ICRE-dependent gene activation, requiring the regulatory genes INO2 and INO4, is repressed in the presence of the phospholipid precursors inositol and choline. INO2 and, to a less extent, INO4 are positively autoregulated by functional ICRE sequences in the respective upstream regions. However, an INO2 allele devoid of its ICRE functionally complemented an ino2 mutation and completely restored inositol/choline regulation of Ino2p-dependent reporter genes. Low-level expression of INO2 and INO4 genes, each under control of the heterologous MET25 promoter, did not alter the regulatory pattern of target genes. Thus, upstream regions of INO2 and INO4 are not crucial for transcriptional control of ICRE-dependent genes by inositol and choline. Interestingly, over-expression of INO2, but not of INO4, counteracted repression by phospholipid precursors. Possibly, a functional antagonism between INO2 and a negative regulator is the key event responsible for repression or de-repression.

Sirt1 suppresses RNA synthesis after UV irradiation in combined xeroderma pigmentosum group D/Cockayne syndrome (XP-D/CS) cells.

PubMed

Vélez-Cruz, Renier; Zadorin, Anton S; Coin, Frédéric; Egly, Jean-Marc

2013-01-15

Specific mutations in the XPD subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with "pure" XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/CS that cannot be explained by a DNA repair defect.

The role of LANP and ataxin 1 in E4F-mediated transcriptional repression

PubMed Central

Cvetanovic, Marija; Rooney, Robert J; Garcia, Jesus J; Toporovskaya, Nataliya; Zoghbi, Huda Y; Opal, Puneet

2007-01-01

The leucine-rich acidic nuclear protein (LANP) belongs to the INHAT family of corepressors that inhibits histone acetyltransferases. The mechanism by which LANP restricts its repression to specific genes is unknown. Here, we report that LANP forms a complex with transcriptional repressor E4F and modulates its activity. As LANP interacts with ataxin 1—a protein mutated in the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1)—we tested whether ataxin 1 can alter the E4F–LANP interaction. We show that ataxin 1 relieves the transcriptional repression induced by the LANP–E4F complex by competing with E4F for LANP. These results provide the first functional link, to our knowledge, between LANP and ataxin 1, and indicate a potential mechanism for the transcriptional aberrations observed in SCA1. PMID:17557114

Corepressors: custom tailoring and alterations while you wait

PubMed Central

Goodson, Michael; Jonas, Brian A.; Privalsky, Martin A.

2005-01-01

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages. PMID:16604171

Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism in Bacillus subtilis Requires Spx

PubMed Central

Erwin, Kyle N.; Nakano, Shunji; Zuber, Peter

2005-01-01

Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions. PMID:15937167

MSX1 cooperates with histone H1b for inhibition of transcription and myogenesis.

PubMed

Lee, Hansol; Habas, Raymond; Abate-Shen, Cory

2004-06-11

During embryogenesis, differentiation of skeletal muscle is regulated by transcription factors that include members of the Msx homeoprotein family. By investigating Msx1 function in repression of myogenic gene expression, we identified a physical interaction between Msx1 and H1b, a specific isoform of mouse histone H1. We found that Msx1 and H1b bind to a key regulatory element of MyoD, a central regulator of skeletal muscle differentiation, where they induce repressed chromatin. Moreover, Msx1 and H1b cooperate to inhibit muscle differentiation in cell culture and in Xenopus animal caps. Our findings define a previously unknown function for "linker" histones in gene-specific transcriptional regulation.

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.

BEND3 mediates transcriptional repression and heterochromatin organization

PubMed Central

Khan, Abid; Prasanth, Supriya G

2015-01-01

Transcription repression plays a central role in gene regulation. Transcription repressors utilize diverse strategies to mediate transcriptional repression. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein represses rDNA transcription by stabilizing a NoRC component. We discuss the role of BEND3 as a global regulator of gene expression and propose a model whereby BEND3 associates with chromatin remodeling complexes to modulate gene expression and heterochromatin organization. PMID:26507581

BEND3 mediates transcriptional repression and heterochromatin organization.

PubMed

Khan, Abid; Prasanth, Supriya G

2015-01-01

Transcription repression plays a central role in gene regulation. Transcription repressors utilize diverse strategies to mediate transcriptional repression. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein represses rDNA transcription by stabilizing a NoRC component. We discuss the role of BEND3 as a global regulator of gene expression and propose a model whereby BEND3 associates with chromatin remodeling complexes to modulate gene expression and heterochromatin organization.

Identification of the gene transcription repressor domain of Gli3.

PubMed

Tsanev, Robert; Tiigimägi, Piret; Michelson, Piret; Metsis, Madis; Østerlund, Torben; Kogerman, Priit

2009-01-05

Gli transcription factors are downstream targets of the Hedgehog signaling pathway. Two of the three Gli proteins harbor gene transcription repressor function in the N-terminal half. We have analyzed the sequences and identified a potential repressor domain in Gli2 and Gli3 and have tested this experimentally. Overexpression studies confirm that the N-terminal parts harbor gene repression activity and we mapped the minimal repressor to residues 106 till 236 in Gli3. Unlike other mechanisms that inhibit Gli induced gene transcription, the repressor domain identified here does not utilize Histone deacetylases (HDACs) to achieve repression, as confirmed by HDAC inhibition studies and pull-down assays. This distinguishes the identified domain from other regulatory parts with negative influence on transcription.

The influence of repressor DNA binding site architecture on transcriptional control.

PubMed

Park, Dan M; Kiley, Patricia J

2014-08-26

How the architecture of DNA binding sites dictates the extent of repression of promoters is not well understood. Here, we addressed the importance of the number and information content of the three direct repeats (DRs) in the binding and repression of the icdA promoter by the phosphorylated form of the global Escherichia coli repressor ArcA (ArcA-P). We show that decreasing the information content of the two sites with the highest information (DR1 and DR2) eliminated ArcA binding to all three DRs and ArcA repression of icdA. Unexpectedly, we also found that DR3 occupancy functions principally in repression, since mutation of this low-information-content site both eliminated DNA binding to DR3 and significantly weakened icdA repression, despite the fact that binding to DR1 and DR2 was intact. In addition, increasing the information content of any one of the three DRs or addition of a fourth DR increased ArcA-dependent repression but perturbed signal-dependent regulation of repression. Thus, our data show that the information content and number of DR elements are critical architectural features for maintaining a balance between high-affinity binding and signal-dependent regulation of icdA promoter function in response to changes in ArcA-P levels. Optimization of such architectural features may be a common strategy to either dampen or enhance the sensitivity of DNA binding among the members of the large OmpR/PhoB family of regulators as well as other transcription factors. In Escherichia coli, the response regulator ArcA maintains homeostasis of redox carriers under O2-limiting conditions through a comprehensive repression of carbon oxidation pathways that require aerobic respiration to recycle redox carriers. Although a binding site architecture comprised of a variable number of sequence recognition elements has been identified within the promoter regions of ArcA-repressed operons, it is unclear how this variable architecture dictates transcriptional regulation. By dissecting the role of multiple sequence elements within the icdA promoter, we provide insight into the design principles that allow ArcA to repress transcription within diverse promoter contexts. Our data suggest that the arrangement of recognition elements is tailored to achieve sufficient repression of a given promoter while maintaining appropriate signal-dependent regulation of repression, providing insight into how diverse binding site architectures link changes in O2 with the fine-tuning of carbon oxidation pathway levels. Copyright © 2014 Park and Kiley.

Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot.

PubMed Central

Hirota, Kouji; Hoffman, Charles S; Shibata, Takehiko; Ohta, Kunihiro

2003-01-01

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1(+) gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function. PMID:14573465

WRKY transcription factors.

PubMed

Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J

2010-05-01

WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy. 2010 Elsevier Ltd. All rights reserved.

GRG5/AES interacts with T-cell factor 4 (TCF4) and downregulates Wnt signaling in human cells and zebrafish embryos.

PubMed

Costa, Angela M Sousa; Pereira-Castro, Isabel; Ricardo, Elisabete; Spencer, Forrest; Fisher, Shannon; da Costa, Luís Teixeira

2013-01-01

Transcriptional control by TCF/LEF proteins is crucial in key developmental processes such as embryo polarity, tissue architecture and cell fate determination. TCFs associate with β-catenin to activate transcription in the presence of Wnt signaling, but in its absence act as repressors together with Groucho-family proteins (GRGs). TCF4 is critical in vertebrate intestinal epithelium, where TCF4-β-catenin complexes are necessary for the maintenance of a proliferative compartment, and their abnormal formation initiates tumorigenesis. However, the extent of TCF4-GRG complexes' roles in development and the mechanisms by which they repress transcription are not completely understood. Here we characterize the interaction between TCF4 and GRG5/AES, a Groucho family member whose functional relationship with TCFs has been controversial. We map the core GRG interaction region in TCF4 to a 111-amino acid fragment and show that, in contrast to other GRGs, GRG5/AES-binding specifically depends on a 4-amino acid motif (LVPQ) present only in TCF3 and some TCF4 isoforms. We further demonstrate that GRG5/AES represses Wnt-mediated transcription both in human cells and zebrafish embryos. Importantly, we provide the first evidence of an inherent repressive function of GRG5/AES in dorsal-ventral patterning during early zebrafish embryogenesis. These results improve our understanding of TCF-GRG interactions, have significant implications for models of transcriptional repression by TCF-GRG complexes, and lay the groundwork for in depth direct assessment of the potential role of Groucho-family proteins in both normal and abnormal development.

Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense Transcription

PubMed Central

Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J.; van Hooff, Sander; van Leenen, Dik

2012-01-01

Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3′-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms. PMID:23028359

Extensive interactions between HIV TAT and TAF(II)250.

PubMed

Weissman, J D; Hwang, J R; Singer, D S

2001-03-09

The HIV transactivator, Tat, has been shown to be capable of potent repression of transcription initiation. Repression is mediated by the C-terminal segment of Tat, which binds the TFIID component, TAF(II)250, although the site(s) of interaction were not defined previously. We now report that the interaction between Tat and TAF(II)250 is extensive and involves multiple contacts between the Tat protein and TAF(II)250. The C-terminal domain of Tat, which is necessary for repression of transcription initiation, binds to a segment of TAF(II)250 that encompasses its acetyl transferase (AT) domain (885-1034 amino acids (aa)). Surprisingly, the N-terminal segment of Tat, which contains its activation domains, also binds to TAF(II)250 and interacts with two discontinuous segments of TAF(II)250 located between 885 and 984 aa and 1120 and 1279 aa. Binding of Tat to the 885-984 aa segment of TAF(II)250 requires the cysteine-rich domain of Tat, but not the acidic or glutamine-rich domains. Binding by the N-terminal domain of Tat to the 1120-1279 aa TAF(II)250 segment does not involve the acidic, cysteine- or glutamine-rich domains. Repression of transcription initiation by Tat requires functional TAF(II)250. We now demonstrate that transcription of the HIV LTR does not depend on TAF(II)250 which may account for its resistance to Tat mediated repression.

The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing

PubMed Central

Bartok, Osnat; Teesalu, Mari; Ashwall-Fluss, Reut; Pandey, Varun; Hanan, Mor; Rovenko, Bohdana M; Poukkula, Minna; Havula, Essi; Moussaieff, Arieh; Vodala, Sadanand; Nahmias, Yaakov; Kadener, Sebastian; Hietakangas, Ville

2015-01-01

Nutrient sensing pathways adjust metabolism and physiological functions in response to food intake. For example, sugar feeding promotes lipogenesis by activating glycolytic and lipogenic genes through the Mondo/ChREBP-Mlx transcription factor complex. Concomitantly, other metabolic routes are inhibited, but the mechanisms of transcriptional repression upon sugar sensing have remained elusive. Here, we characterize cabut (cbt), a transcription factor responsible for the repressive branch of the sugar sensing transcriptional network in Drosophila. We demonstrate that cbt is rapidly induced upon sugar feeding through direct regulation by Mondo-Mlx. We found that CBT represses several metabolic targets in response to sugar feeding, including both isoforms of phosphoenolpyruvate carboxykinase (pepck). Deregulation of pepck1 (CG17725) in mlx mutants underlies imbalance of glycerol and glucose metabolism as well as developmental lethality. Furthermore, we demonstrate that cbt provides a regulatory link between nutrient sensing and the circadian clock. Specifically, we show that a subset of genes regulated by the circadian clock are also targets of CBT. Moreover, perturbation of CBT levels leads to deregulation of the circadian transcriptome and circadian behavioral patterns. PMID:25916830

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

PubMed

Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

Repressive Chromatin in Caenorhabditis elegans: Establishment, Composition, and Function

PubMed Central

Ahringer, Julie; Gasser, Susan M.

2018-01-01

Chromatin is organized and compacted in the nucleus through the association of histones and other proteins, which together control genomic activity. Two broad types of chromatin can be distinguished: euchromatin, which is generally transcriptionally active, and heterochromatin, which is repressed. Here we examine the current state of our understanding of repressed chromatin in Caenorhabditis elegans, focusing on roles of histone modifications associated with repression, such as methylation of histone H3 lysine 9 (H3K9me2/3) or the Polycomb Repressive Complex 2 (MES-2/3/6)-deposited modification H3K27me3, and on proteins that recognize these modifications. Proteins involved in chromatin repression are important for development, and have demonstrated roles in nuclear organization, repetitive element silencing, genome integrity, and the regulation of euchromatin. Additionally, chromatin factors participate in repression with small RNA pathways. Recent findings shed light on heterochromatin function and regulation in C. elegans, and should inform our understanding of repressed chromatin in other animals. PMID:29378810

RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes

PubMed Central

Rose, Nathan R; King, Hamish W; Blackledge, Neil P; Fursova, Nadezda A; Ember, Katherine JI; Fischer, Roman; Kessler, Benedikt M; Klose, Robert J

2016-01-01

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation. DOI: http://dx.doi.org/10.7554/eLife.18591.001 PMID:27705745

The corepressor CtBP interacts with Evi-1 to repress transforming growth factor beta signaling.

PubMed

Izutsu, K; Kurokawa, M; Imai, Y; Maki, K; Mitani, K; Hirai, H

2001-05-01

Evi-1 is a zinc finger nuclear protein whose inappropriate expression leads to leukemic transformation of hematopoietic cells in mice and humans. This was previously shown to block the antiproliferative effect of transforming growth factor beta (TGF-beta). Evi-1 represses TGF-beta signaling by direct interaction with Smad3 through its first zinc finger motif. Here, it is demonstrated that Evi-1 represses Smad-induced transcription by recruiting C-terminal binding protein (CtBP) as a corepressor. Evi-1 associates with CtBP1 through one of the consensus binding motifs, and this association is required for efficient inhibition of TGF-beta signaling. A specific inhibitor for histone deacetylase (HDAc) alleviates Evi-1-mediated repression of TGF-beta signaling, suggesting that HDAc is involved in the transcriptional repression by Evi-1. This identifies a novel function of Evi-1 as a member of corepressor complexes and suggests that aberrant recruitment of corepressors is one of the mechanisms for Evi-1-induced leukemogenesis.

Separate necdin domains bind ARNT2 and HIF1{alpha} and repress transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedman, Eitan R.; Fan Chenming

2007-11-09

PWS is caused by the loss of expression of a set of maternally imprinted genes including NECDIN (NDN). NDN is expressed in post-mitotic neurons and plays an essential role in PWS as mouse models lacking only the Ndn gene mimic aspects of this disease. Patients haploid for SIM1 develop a PW-like syndrome. Here, we report that NDN directly interacts with ARNT2, a bHLH-PAS protein and dimer partner for SIM1. We also found that NDN can interact with HIF1{alpha}. We showed that NDN can repress transcriptional activation mediated by ARNT2:SIM1 as well as ARNT2:HIF1{alpha}. The N-terminal 115 residues of NDN aremore » sufficient for interaction with the bHLH domains of ARNT2 or HIF1{alpha} but not for transcriptional repression. Using GAL4-NDN fusion proteins, we determined that NDN possesses multiple repression domains. We thus propose that NDN regulates neuronal function and hypoxic response by regulating the activities of the ARNT2:SIM1 and ARNT2:HIF1{alpha} dimers, respectively.« less

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

PubMed Central

Battistelli, C; Cicchini, C; Santangelo, L; Tramontano, A; Grassi, L; Gonzalez, F J; de Nonno, V; Grassi, G; Amicone, L; Tripodi, M

2017-01-01

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT. PMID:27452518

The transcription factor NRSF contributes to epileptogenesis by selective repression of a subset of target genes

PubMed Central

McClelland, Shawn; Brennan, Gary P; Dubé, Celine; Rajpara, Seeta; Iyer, Shruti; Richichi, Cristina; Bernard, Christophe; Baram, Tallie Z

2014-01-01

The mechanisms generating epileptic neuronal networks following insults such as severe seizures are unknown. We have previously shown that interfering with the function of the neuron-restrictive silencer factor (NRSF/REST), an important transcription factor that influences neuronal phenotype, attenuated development of this disorder. In this study, we found that epilepsy-provoking seizures increased the low NRSF levels in mature hippocampus several fold yet surprisingly, provoked repression of only a subset (∼10%) of potential NRSF target genes. Accordingly, the repressed gene-set was rescued when NRSF binding to chromatin was blocked. Unexpectedly, genes selectively repressed by NRSF had mid-range binding frequencies to the repressor, a property that rendered them sensitive to moderate fluctuations of NRSF levels. Genes selectively regulated by NRSF during epileptogenesis coded for ion channels, receptors, and other crucial contributors to neuronal function. Thus, dynamic, selective regulation of NRSF target genes may play a role in influencing neuronal properties in pathological and physiological contexts. DOI: http://dx.doi.org/10.7554/eLife.01267.001 PMID:25117540

Oncogenic mechanisms of Evi-1 protein.

PubMed

Hirai, H; Izutsu, K; Kurokawa, M; Mitani, K

2001-08-01

Although Evi-1 is thought to promote growth or block differentiation in some cell types, its biological functions have not been elucidated. To explore the mechanisms underlying Evi-1-induced oncogenesis, we investigated whether Evi-1 affects the signaling of transforming growth factor beta (TGF-beta), which inhibits proliferation of a wide range of cell types and is one of the most studied growth regulatory factors. We demonstrated that Evi-1 represses TGF-beta signaling and antagonizes its growth-inhibitory effects. Two separate regions of Evi-1 are responsible for this repression, one of which is the first zinc-finger domain. Through this domain, Evi-1 physically interacts with Smad3, an intracellular mediator of TGF-beta signaling, thereby suppressing the transcriptional activity of Smad3. These results define a novel function of Evi-1 as a repressor of signaling components of TGF-beta. We also demonstrated that Evi-1 represses Smad-induced transcriptional activation by recruiting CtBP as a corepressor. Evi-1 associates with CtBP1 through one of the CtBP-binding consensus motifs within the region from amino acid 544 to 607, and this association is required for the efficient inhibition of TGF-beta signaling. A specific histone deacetylase (HDAc) inhibitor, trichostatin A (TSA), alleviates Evi-1-mediated repression of TGF-beta signaling, suggesting that HDAc is involved in transcriptional repression by Evi-1. This identifies a novel function of Evi-1 as a member of corepressor complexes and suggests that aberrant recruitment of corepressors is one of the mechanisms involved in Evi-1-induced leukemogenesis. These results indicate that specific HDAc inhibitors may be useful in the treatment of Evi-1-induced neoplastic tumors, including myeloid leukemias.

Cryptochrome 1 regulates the circadian clock through dynamic interactions with the BMAL1 C-terminus

PubMed Central

Sammons, Patrick J.; Khan, Sanjoy K.; Parsley, Nicole C.; Ramanathan, Chidambaram; Lee, Hsiau-Wei; Liu, Andrew C.; Partch, Carrie L.

2015-01-01

The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK–BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK–BMAL1 to close the negative feedback loop and generate 24-hour timing is not known. Here we show that CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK–BMAL1. Mutations within the TAD that alter affinities for coregulators change the balance of repression and activation to consequently change intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators and highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing. PMID:25961797

Epigenetic repression of the Igk locus by STAT5-mediated Ezh2 recruitment

PubMed Central

Mandal, Malay; Powers, Sarah E.; Maienschein-Cline, Mark; Bartom, Elizabeth T.; Hamel, Keith M.; Kee, Barbara L.; Dinner, Aaron R.; Clark, Marcus R.

2011-01-01

During B lymphopoiesis, Igk recombination requires pre-B cell receptor (pre-BCR) expression and escape from interleukin 7 receptor (IL-7R) signaling. By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that STAT5 tetramer bound the Igk intronic enhancer (Eκi), leading to recruitment of the histone methyltransferase Ezh2. Ezh2 marked H3K27me3 throughout Jκ to Cκ. In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R–STAT5 signaling and E2A bound Eκi, resulting in acquisition of H3K4me1 and H4Ac. Genome-wide analyses revealed a STAT5 tetrameric binding motif associated with transcriptional repression. These data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression. PMID:22037603

p21 as a Transcriptional Co-Repressor of S-Phase and Mitotic Control Genes

PubMed Central

Ferrándiz, Nuria; Caraballo, Juan M.; García-Gutierrez, Lucía; Devgan, Vikram; Rodriguez-Paredes, Manuel; Lafita, M. Carmen; Bretones, Gabriel; Quintanilla, Andrea; Muñoz-Alonso, M. Jose; Blanco, Rosa; Reyes, Jose C.; Agell, Neus; Delgado, M. Dolores; Dotto, G. Paolo; León, Javier

2012-01-01

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes. PMID:22662213

A signaling role of histone-binding proteins and INHAT subunits pp32 and Set/TAF-Ibeta in integrating chromatin hypoacetylation and transcriptional repression.

PubMed

Kutney, Sara N; Hong, Rui; Macfarlan, Todd; Chakravarti, Debabrata

2004-07-16

Various post-translational modifications of histones significantly influence gene transcription. Although un- or hypoacetylated histones are tightly linked to transcriptional repression, the mechanisms and identities of chromatin signal transducer proteins integrating histone hypoacetylation into repression in humans have remained largely unknown. Here we show that the mammalian histone-binding proteins and inhibitor of acetyltransferases (INHAT) complex subunits, Set/template-activating factor-Ibeta (TAF-Ibeta) and pp32, specifically bind to unacetylated, hypoacetylated, and repressively marked histones but not to hyperacetylated histones. Additionally, Set/TAF-Ibeta and pp32 associate with histone deacetylases in vitro and in vivo and repress transcription from a chromatin-integrated template in vivo. Finally, Set/TAF-Ibeta and pp32 associate with an endogenous estrogen receptor-regulated gene, EB1, in the hypoacetylated transcriptionally inactive state but not with the hyperacetylated transcriptionally active form. Together, these data define a novel in vivo mechanistic role for the mammalian Set/TAF-Ibeta and pp32 proteins as transducers of chromatin signaling by integrating chromatin hypoacetylation and transcriptional repression.

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma

PubMed Central

Gilmore, Sarah A.; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-01-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature. PMID:26177267

Genome-Wide Reprogramming of Transcript Architecture by Temperature Specifies the Developmental States of the Human Pathogen Histoplasma.

PubMed

Gilmore, Sarah A; Voorhies, Mark; Gebhart, Dana; Sil, Anita

2015-07-01

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5' leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature.

GRG5/AES Interacts with T-Cell Factor 4 (TCF4) and Downregulates Wnt Signaling in Human Cells and Zebrafish Embryos

PubMed Central

Costa, Ângela M. Sousa; Pereira-Castro, Isabel; Ricardo, Elisabete; Spencer, Forrest; Fisher, Shannon; da Costa, Luís Teixeira

2013-01-01

Transcriptional control by TCF/LEF proteins is crucial in key developmental processes such as embryo polarity, tissue architecture and cell fate determination. TCFs associate with β-catenin to activate transcription in the presence of Wnt signaling, but in its absence act as repressors together with Groucho-family proteins (GRGs). TCF4 is critical in vertebrate intestinal epithelium, where TCF4-β-catenin complexes are necessary for the maintenance of a proliferative compartment, and their abnormal formation initiates tumorigenesis. However, the extent of TCF4-GRG complexes’ roles in development and the mechanisms by which they repress transcription are not completely understood. Here we characterize the interaction between TCF4 and GRG5/AES, a Groucho family member whose functional relationship with TCFs has been controversial. We map the core GRG interaction region in TCF4 to a 111-amino acid fragment and show that, in contrast to other GRGs, GRG5/AES-binding specifically depends on a 4-amino acid motif (LVPQ) present only in TCF3 and some TCF4 isoforms. We further demonstrate that GRG5/AES represses Wnt-mediated transcription both in human cells and zebrafish embryos. Importantly, we provide the first evidence of an inherent repressive function of GRG5/AES in dorsal-ventral patterning during early zebrafish embryogenesis. These results improve our understanding of TCF-GRG interactions, have significant implications for models of transcriptional repression by TCF-GRG complexes, and lay the groundwork for in depth direct assessment of the potential role of Groucho-family proteins in both normal and abnormal development. PMID:23840876

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.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP.

PubMed

Pagan, Julia K; Arnold, Jeremy; Hanchard, Kim J; Kumar, Raman; Bruno, Tiziana; Jones, Mathew J K; Richard, Derek J; Forrest, Alistair; Spurdle, Amanda; Verdin, Eric; Crossley, Merlin; Fanciulli, Maurizio; Chenevix-Trench, Georgia; Young, David B; Khanna, Kum Kum

2007-05-18

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

Dampening DNA binding: a common mechanism of transcriptional repression for both ncRNAs and protein domains.

PubMed

Goodrich, James A; Kugel, Jennifer F

2010-01-01

With eukaryotic non-coding RNAs (ncRNAs) now established as critical regulators of cellular transcription, the true diversity with which they can elicit biological effects is beginning to be appreciated. Two ncRNAs, mouse B2 RNA and human Alu RNA, have been found to repress mRNA transcription in response to heat shock. They do so by binding directly to RNA polymerase II, assembling into complexes on promoter DNA, and disrupting contacts between the polymerase and the DNA. Such a mechanism of repression had not previously been observed for a eukaryotic ncRNA; however, there are examples of eukaryotic protein domains that repress transcription by blocking essential protein-DNA interactions. Comparing the mechanism of transcriptional repression utilized by these protein domains to that used by B2 and Alu RNAs raises intriguing questions regarding transcriptional control, and how B2 and Alu RNAs might themselves be regulated.

Identification of HDA15-PIF1 as a key repression module directing the transcriptional network of seed germination in the dark.

PubMed

Gu, Dachuan; Chen, Chia-Yang; Zhao, Minglei; Zhao, Linmao; Duan, Xuewu; Duan, Jun; Wu, Keqiang; Liu, Xuncheng

2017-07-07

Light is a major external factor in regulating seed germination. Photoreceptor phytochrome B (PHYB) plays a predominant role in promoting seed germination in the initial phase after imbibition, partially by repressing phytochrome-interacting factor1 (PIF1). However, the mechanism underlying the PHYB-PIF1-mediated transcription regulation remains largely unclear. Here, we identified that histone deacetylase15 (HDA15) is a negative component of PHYB-dependent seed germination. Overexpression of HDA15 in Arabidopsis inhibits PHYB-dependent seed germination, whereas loss of function of HDA15 increases PHYB-dependent seed germination. Genetic evidence indicated that HDA15 acts downstream of PHYB and represses seed germination dependent on PIF1. Furthermore, HDA15 interacts with PIF1 both in vitro and in vivo. Genome-wide transcriptome analysis revealed that HDA15 and PIF1 co-regulate the transcription of the light-responsive genes involved in multiple hormonal signaling pathways and cellular processes in germinating seeds in the dark. In addition, PIF1 recruits HDA15 to the promoter regions of target genes and represses their expression by decreasing the histone H3 acetylation levels in the dark. Taken together, our analysis uncovered the role of histone deacetylation in the light-regulated seed germination process and identified that HDA15-PIF1 acts as a key repression module directing the transcription network of seed germination. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Identification of HDA15-PIF1 as a key repression module directing the transcriptional network of seed germination in the dark

PubMed Central

Gu, Dachuan; Chen, Chia-Yang; Zhao, Minglei; Zhao, Linmao; Duan, Xuewu

2017-01-01

Abstract Light is a major external factor in regulating seed germination. Photoreceptor phytochrome B (PHYB) plays a predominant role in promoting seed germination in the initial phase after imbibition, partially by repressing phytochrome-interacting factor1 (PIF1). However, the mechanism underlying the PHYB-PIF1-mediated transcription regulation remains largely unclear. Here, we identified that histone deacetylase15 (HDA15) is a negative component of PHYB-dependent seed germination. Overexpression of HDA15 in Arabidopsis inhibits PHYB-dependent seed germination, whereas loss of function of HDA15 increases PHYB-dependent seed germination. Genetic evidence indicated that HDA15 acts downstream of PHYB and represses seed germination dependent on PIF1. Furthermore, HDA15 interacts with PIF1 both in vitro and in vivo. Genome-wide transcriptome analysis revealed that HDA15 and PIF1 co-regulate the transcription of the light-responsive genes involved in multiple hormonal signaling pathways and cellular processes in germinating seeds in the dark. In addition, PIF1 recruits HDA15 to the promoter regions of target genes and represses their expression by decreasing the histone H3 acetylation levels in the dark. Taken together, our analysis uncovered the role of histone deacetylation in the light-regulated seed germination process and identified that HDA15-PIF1 acts as a key repression module directing the transcription network of seed germination. PMID:28444370

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type β transforming growth factor

PubMed Central

Xu, Weidong; Angelis, Konstantina; Danielpour, David; Haddad, Maher M.; Bischof, Oliver; Campisi, Judith; Stavnezer, Ed; Medrano, Estela E.

2000-01-01

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type β transforming growth factor (TGF-β) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-β. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-β-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-β-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-β-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-β-mediated growth inhibition in a prostate-derived epithelial cell line. PMID:10811875

Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor.

PubMed

Xu, W; Angelis, K; Danielpour, D; Haddad, M M; Bischof, O; Campisi, J; Stavnezer, E; Medrano, E E

2000-05-23

The c-ski protooncogene encodes a transcription factor that binds DNA only in association with other proteins. To identify co-binding proteins, we performed a yeast two-hybrid screen. The results of the screen and subsequent co-immunoprecipitation studies identified Smad2 and Smad3, two transcriptional activators that mediate the type beta transforming growth factor (TGF-beta) response, as Ski-interacting proteins. In Ski-transformed cells, all of the Ski protein was found in Smad3-containing complexes that accumulated in the nucleus in the absence of added TGF-beta. DNA binding assays showed that Ski, Smad2, Smad3, and Smad4 form a complex with the Smad/Ski binding element GTCTAGAC (SBE). Ski repressed TGF-beta-induced expression of 3TP-Lux, the natural plasminogen activator inhibitor 1 promoter and of reporter genes driven by the SBE and the related CAGA element. In addition, Ski repressed a TGF-beta-inducible promoter containing AP-1 (TRE) elements activated by a combination of Smads, Fos, and/or Jun proteins. Ski also repressed synergistic activation of promoters by combinations of Smad proteins but failed to repress in the absence of Smad4. Thus, Ski acts in opposition to TGF-beta-induced transcriptional activation by functioning as a Smad-dependent co-repressor. The biological relevance of this transcriptional repression was established by showing that overexpression of Ski abolished TGF-beta-mediated growth inhibition in a prostate-derived epithelial cell line.

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

PubMed

Born, Nadine; Thiesen, Hans-Jürgen; Lorenz, Peter

2014-01-01

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

Cloning and functional analysis of the promoter region of the human Disc large gene.

PubMed

Cavatorta, Ana Laura; Giri, Adriana A; Banks, Lawrence; Gardiol, Daniela

2008-11-15

A number of studies have demonstrated the involvement of human Disc large (DLG1) in the control of both cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 transcription are not fully understood. This is relevant since DLG1 is lost in many tumours during the later stages of malignant progression. Therefore, we performed the cloning and functional analysis of a genomic 5' flanking region of the DLG1 open reading frame with promoter activity. We analyzed the activity of a series of 5' deletion constructs of the DLG1 promoter and determined the minimal essential sequences that are required for promoter activity as well as cis-elements that regulate transcription. We found, within the DLG1 promoter sequences, consensus-binding sites for the Snail family of transcription factors that repress the expression of epithelial markers and are up-regulated in a variety of tumours. Snail transcription factors repress the transcriptional activity of the DLG1 promoter and, ectopically expressed Snail proteins bind to the native DLG1 promoter. These data suggest a role for Snail transcription factors in the control of DLG1 expression and provide a basis for understanding the transcriptional regulation of DLG1.

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

PubMed Central

Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674

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

The C2H2 Transcription Factor REGULATOR OF SYMBIOSOME DIFFERENTIATION Represses Transcription of the Secretory Pathway Gene VAMP721a and Promotes Symbiosome Development in Medicago truncatula[W][OPEN

PubMed Central

Sinharoy, Senjuti; Torres-Jerez, Ivone; Bandyopadhyay, Kaustav; Kereszt, Attila; Pislariu, Catalina I.; Nakashima, Jin; Benedito, Vagner A.; Kondorosi, Eva; Udvardi, Michael K.

2013-01-01

Transcription factors (TFs) are thought to regulate many aspects of nodule and symbiosis development in legumes, although few TFs have been characterized functionally. Here, we describe REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD) of Medicago truncatula, a member of the Cysteine-2/Histidine-2 (C2H2) family of plant TFs that is required for normal symbiosome differentiation during nodule development. RSD is expressed in a nodule-specific manner, with maximal transcript levels in the bacterial invasion zone. A tobacco (Nicotiana tabacum) retrotransposon (Tnt1) insertion rsd mutant produced nodules that were unable to fix nitrogen and that contained incompletely differentiated symbiosomes and bacteroids. RSD protein was localized to the nucleus, consistent with a role of the protein in transcriptional regulation. RSD acted as a transcriptional repressor in a heterologous yeast assay. Transcriptome analysis of an rsd mutant identified 11 genes as potential targets of RSD repression. RSD interacted physically with the promoter of one of these genes, VAMP721a, which encodes vesicle-associated membrane protein 721a. Thus, RSD may influence symbiosome development in part by repressing transcription of VAMP721a and modifying vesicle trafficking in nodule cells. This establishes RSD as a TF implicated directly in symbiosome and bacteroid differentiation and a transcriptional regulator of secretory pathway genes in plants. PMID:24082011

The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula.

PubMed

Sinharoy, Senjuti; Torres-Jerez, Ivone; Bandyopadhyay, Kaustav; Kereszt, Attila; Pislariu, Catalina I; Nakashima, Jin; Benedito, Vagner A; Kondorosi, Eva; Udvardi, Michael K

2013-09-01

Transcription factors (TFs) are thought to regulate many aspects of nodule and symbiosis development in legumes, although few TFs have been characterized functionally. Here, we describe regulator of symbiosome differentiation (RSD) of Medicago truncatula, a member of the Cysteine-2/Histidine-2 (C2H2) family of plant TFs that is required for normal symbiosome differentiation during nodule development. RSD is expressed in a nodule-specific manner, with maximal transcript levels in the bacterial invasion zone. A tobacco (Nicotiana tabacum) retrotransposon (Tnt1) insertion rsd mutant produced nodules that were unable to fix nitrogen and that contained incompletely differentiated symbiosomes and bacteroids. RSD protein was localized to the nucleus, consistent with a role of the protein in transcriptional regulation. RSD acted as a transcriptional repressor in a heterologous yeast assay. Transcriptome analysis of an rsd mutant identified 11 genes as potential targets of RSD repression. RSD interacted physically with the promoter of one of these genes, VAMP721a, which encodes vesicle-associated membrane protein 721a. Thus, RSD may influence symbiosome development in part by repressing transcription of VAMP721a and modifying vesicle trafficking in nodule cells. This establishes RSD as a TF implicated directly in symbiosome and bacteroid differentiation and a transcriptional regulator of secretory pathway genes in plants.

Developmental control of transcriptional and proliferative potency during the evolutionary emergence of animals

PubMed Central

Arenas-Mena, Cesar; Coffman, James A.

2016-01-01

Summary It is proposed that the evolution of complex animals required repressive genetic mechanisms for controlling the transcriptional and proliferative potency of cells. Unicellular organisms are transcriptionally potent, able to express their full genetic complement as the need arises through their life cycle, whereas differentiated cells of multicellular organisms can only express a fraction of their genomic potential. Likewise, whereas cell proliferation in unicellular organisms is primarily limited by nutrient availability, cell proliferation in multicellular organisms is developmentally regulated. Repressive genetic controls limiting the potency of cells at the end of ontogeny would have stabilized the gene expression states of differentiated cells and prevented disruptive proliferation, allowing the emergence of diverse cell types and functional shapes. We propose that distal cis-regulatory elements represent the primary innovations that set the stage for the evolution of developmental gene regulatory networks and the repressive control of key multipotency and cell-cycle control genes. The testable prediction of this model is that the genomes of extant animals, unlike those of our unicellular relatives, encode gene regulatory circuits dedicated to the developmental control of transcriptional and proliferative potency. PMID:26173445

E-box-independent regulation of transcription and differentiation by MYC.

PubMed

Uribesalgo, Iris; Buschbeck, Marcus; Gutiérrez, Arantxa; Teichmann, Sophia; Demajo, Santiago; Kuebler, Bernd; Nomdedéu, Josep F; Martín-Caballero, Juan; Roma, Guglielmo; Benitah, Salvador Aznar; Di Croce, Luciano

2011-10-23

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.

Transcriptional repression of epithelial cell adhesion molecule (EpCAM) contributes to p53 control of breast cancer invasion

PubMed Central

Sankpal, NV; Willman, MW; Fleming, TP; Mayfield, J; Gillanders, WE

2014-01-01

p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis and the maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We demonstrate by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function, and loss-of-function experimental systems. Induction of wildtype p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (1) wildtype p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (2) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion. PMID:19141643

EZH2: a pivotal regulator in controlling cell differentiation.

PubMed

Chen, Ya-Huey; Hung, Mien-Chie; Li, Long-Yuan

2012-01-01

Epigenetic regulation plays an important role in stem cell self-renewal, maintenance and lineage differentiation. The epigenetic profiles of stem cells are related to their transcriptional signature. Enhancer of Zeste homlog 2 (EZH2), a catalytic subunit of epigenetic regulator Polycomb repressive complex 2 (PRC2), has been shown to be a key regulator in controlling cellular differentiation. EZH2 is a histone methyltransferase that not only methylates histone H3 on Lys 27 (H3K27me3) but also interacts with and recruits DNA methyltransferases to methylate CpG at certain EZH2 target genes to establish firm repressive chromatin structures, contributing to tumor progression and the regulation of development and lineage commitment both in embryonic stem cells (ESCs) and adult stem cells. In addition to its well-recognized epigenetic gene silencing function, EZH2 also directly methylates nonhistone targets such as the cardiac transcription factor, GATA4, resulting in attenuated GATA4 transcriptional activity and gene repression. This review addresses recent progress toward the understanding of the biological functions and regulatory mechanisms of EZH2 and its targets as well as their roles in stem cell maintenance and cell differentiation.

Ski can negatively regulates macrophage differentiation through its interaction with PU.1

PubMed Central

Ueki, N; Zhang, L; Haymann, MJ

2010-01-01

In the hematopoietic cell system, the oncoprotein Ski dramatically affects growth and differentiation programs, in some cases leading to malignant leukemia. However, little is known about the interaction partners or signaling pathways involved in the Ski-mediated block of differentiation in hematopoietic cells. Here we show that Ski interacts with PU.1, a lineage-specific transcription factor essential for terminal myeloid differentiation, and thereby represses PU.1-dependent transcriptional activation. Consistent with this, Ski inhibits the biological function of PU.1 to promote myeloid cells to differentiate into macrophage colony-stimulating factor receptor (M-CSFR)-positive macrophages. Using a Ski mutant deficient in PU.1 binding, we demonstrate that Ski–PU.1 interaction is critical for Ski's ability to repress PU.1-dependent transcription and block macrophage differentiation. Furthermore, we provide evidence that Ski-mediated repression of PU.1 is due to Ski's ability to recruit histone deacetylase 3 to PU.1 bound to DNA. Since inactivation of PU.1 is closely related to the development of myeloid leukemia and Ski strongly inhibits PU.1 function, we propose that aberrant Ski expression in certain types of myeloid cell lineages might contribute to leukemogenesis. PMID:17621263

p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

PubMed

Ogden, S K; Lee, K C; Wernke-Dollries, K; Stratton, S A; Aronow, B; Barton, M C

2001-11-09

Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

PubMed

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

The N-CoR complex enables chromatin remodeler SNF2H to enhance repression by thyroid hormone receptor

PubMed Central

Alenghat, Theresa; Yu, Jiujiu; Lazar, Mitchell A

2006-01-01

Unliganded thyroid hormone receptor (TR) actively represses transcription via the nuclear receptor corepressor (N-CoR)/histone deacetylase 3 (HDAC3) complex. Although transcriptional activation by liganded receptors involves chromatin remodeling, the role of ATP-dependent remodeling in receptor-mediated repression is unknown. Here we report that SNF2H, the mammalian ISWI chromatin remodeling ATPase, is critical for repression of a genomically integrated, TR-regulated reporter gene. N-CoR and HDAC3 are both required for recruitment of SNF2H to the repressed gene. SNF2H does not interact directly with the N-CoR/HDAC3 complex, but binds to unacetylated histone H4 tails, suggesting that deacetylase activity of the corepressor complex is critical to SNF2H function. Indeed, HDAC3 as well as SNF2H are required for nucleosomal organization on the TR target gene. Consistent with these findings, reduction of SNF2H induces expression of an endogenous TR-regulated gene, dio1, in liver cells. Thus, although not apparent from studies of transiently transfected reporter genes, gene repression by TR involves the targeting of chromatin remodeling factors to repressed genes by the HDAC activity of nuclear receptor corepressors. PMID:16917504

Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons

DOE PAGES

Sandberg, Magnus; Flandin, Pierre; Silberberg, Shanni; ...

2016-09-21

The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissivemore » chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach gives a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.« less

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Eun Hee; Gorman, Amanda A.; Singh, Puja

2015-12-04

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that ismore » impaired in the HNF1α mutation-driven diabetes. - Highlights: • We identified AES as a transcriptional repressor for HNF1α in pancreatic beta-cell. • AES's repressive activity was HNF1α-specific and was not observed with HNF1β. • AES interacts with the transactivation domain of HNF1α. • Small molecules can be designed or discovered to disrupt this interaction and improve insulin secretion and glucose homeostasis.« less

The Hippo effector Yorkie controls normal tissue growth by antagonizing scalloped-mediated default repression.

PubMed

Koontz, Laura M; Liu-Chittenden, Yi; Yin, Feng; Zheng, Yonggang; Yu, Jianzhong; Huang, Bo; Chen, Qian; Wu, Shian; Pan, Duojia

2013-05-28

The Hippo tumor suppressor pathway restricts tissue growth by inactivating the transcriptional coactivator Yki. Although Sd has been implicated as a DNA-binding transcription factor partner for Yki and can genetically account for gain-of-function Yki phenotypes, how Yki regulates normal tissue growth remains a long-standing puzzle because Sd, unlike Yki, is dispensable for normal growth in most Drosophila tissues. Here we show that the yki mutant phenotypes in multiple developmental contexts are rescued by inactivation of Sd, suggesting that Sd functions as a default repressor and that Yki promotes normal tissue growth by relieving Sd-mediated default repression. We further identify Tgi as a cofactor involved in Sd's default repressor function and demonstrate that the mammalian ortholog of Tgi potently suppresses the YAP oncoprotein in transgenic mice. These findings fill a major gap in Hippo-mediated transcriptional regulation and open up possibilities for modulating the YAP oncoprotein in cancer and regenerative medicine. Copyright © 2013 Elsevier Inc. All rights reserved.

The use of in vitro transcription to probe regulatory functions of viral protein domains.

PubMed

Loewenstein, Paul M; Song, Chao-Zhong; Green, Maurice

2007-01-01

Adenoviruses (Ads), like other DNA tumor viruses, have evolved specific regulatory genes that facilitate virus replication by controlling the transcription of other viral genes as well as that of key cellular genes. In this regard, the E1A transcription unit contains multiple protein domains that can transcriptionally activate or repress cellular genes involved in the regulation of cell proliferation and cell differentiation. Studies using in vitro transcription have provided a basis for a molecular understanding of the interaction of viral regulatory proteins with the transcriptional machinery of the cell and continue to inform our understanding of transcription regulation. This chapter provides examples of the use of in vitro transcription to analyze transcriptional activation and transcriptional repression by purified, recombinant Ad E1A protein domains and single amino acid substitution mutants as well as the use of protein-affinity chromatography to identify host cell transcription factors involved in viral transcriptional regulation. A detailed description is provided of the methodology to prepare nuclear transcription extract, to prepare biologically active protein domains, to prepare affinity depleted transcription extracts, and to analyze transcription by primer extension and by run-off assay using naked DNA templates.

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its rolemore » in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishida, Tamotsu, E-mail: nishida@gene.mie-u.ac.jp; Yamada, Yoshiji

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 inmore » 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. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.« less

Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation.

PubMed

Lorenz, P; Koczan, D; Thiesen, H J

2001-04-01

The KRAB domain of human Kox1, a member of the KRAB C2H2 zinc finger family, confers strong transcriptional repressor activities even to remote promoter positions. Here, HDAC inhibitors were used to demonstrate that histone deacetylation is not required for mediating transcriptional repression of KRAB zinc finger proteins. Two reporter systems with either stably integrated or transiently transfected templates, both under control of strong viral promoters, were analyzed. Under all circumstances, HDAC inhibition did not alter the repression potential of the KRAB domain. In case of the stably integrated luciferase reporter gene system, neither expression levels of the KRAB fusion protein nor complex formation with its putative co-repressor TIF1beta were significantly changed. Furthermore, the TIF1beta/KRAB complex was devoid of mSin3A and HDAC1. In the transient transfection system, the transcriptional repression induced by TIF1beta and HP1alpha was not diminished by HDAC inhibitors, whereas the repressory activity of TIF1alpha was significantly affected. Thus, KRAB, TIF1beta and HP1alpha are likely to be functionally linked. In conclusion, HDAC activity is not essential for the strong transcriptional repressor activity mediated by the KRAB domain of Kox1 in particular and, presumably, by KRAB domains in general. This feature might be helpful in identifying and characterizing target genes under the control of

Thermodynamics-Based Models of Transcriptional Regulation by Enhancers: The Roles of Synergistic Activation, Cooperative Binding and Short-Range Repression

PubMed Central

He, Xin; Samee, Md. Abul Hassan; Blatti, Charles; Sinha, Saurabh

2010-01-01

Quantitative models of cis-regulatory activity have the potential to improve our mechanistic understanding of transcriptional regulation. However, the few models available today have been based on simplistic assumptions about the sequences being modeled, or heuristic approximations of the underlying regulatory mechanisms. We have developed a thermodynamics-based model to predict gene expression driven by any DNA sequence, as a function of transcription factor concentrations and their DNA-binding specificities. It uses statistical thermodynamics theory to model not only protein-DNA interaction, but also the effect of DNA-bound activators and repressors on gene expression. In addition, the model incorporates mechanistic features such as synergistic effect of multiple activators, short range repression, and cooperativity in transcription factor-DNA binding, allowing us to systematically evaluate the significance of these features in the context of available expression data. Using this model on segmentation-related enhancers in Drosophila, we find that transcriptional synergy due to simultaneous action of multiple activators helps explain the data beyond what can be explained by cooperative DNA-binding alone. We find clear support for the phenomenon of short-range repression, where repressors do not directly interact with the basal transcriptional machinery. We also find that the binding sites contributing to an enhancer's function may not be conserved during evolution, and a noticeable fraction of these undergo lineage-specific changes. Our implementation of the model, called GEMSTAT, is the first publicly available program for simultaneously modeling the regulatory activities of a given set of sequences. PMID:20862354

Functional regulation of Q by microRNA172 and transcriptional co-repressor TOPLESS in controlling bread wheat spikelet density.

PubMed

Liu, Pan; Liu, Jie; Dong, Huixue; Sun, Jiaqiang

2018-02-01

Bread wheat (Triticum aestivum) spike architecture is an important agronomic trait. The Q gene plays a key role in the domestication of bread wheat spike architecture. However, the regulatory mechanisms of Q expression and transcriptional activity remain largely unknown. In this study, we show that overexpression of bread wheat tae-miR172 caused a speltoid-like spike phenotype, reminiscent of that in wheat plants with the q gene. The reduction in Q transcript levels in the tae-miR172 overexpression transgenic bread wheat lines suggests that the Q expression can be suppressed by tae-miR172 in bread wheat. Indeed, our RACE analyses confirmed that the Q mRNA is targeted by tae-miR172 for cleavage. According to our analyses, the Q protein is localized in nucleus and confers transcriptional repression activity. Meanwhile, the Q protein could physically interact with the bread wheat transcriptional co-repressor TOPLESS (TaTPL). Specifically, the N-terminal ethylene-responsive element binding factor-associated amphiphilic repression (EAR) (LDLNVE) motif but not the C-terminal EAR (LDLDLR) motif of Q protein mediates its interaction with the CTLH motif of TaTPL. Moreover, we show that the N-terminal EAR motif of Q protein is also essentially required for the transcriptional repression activity of Q protein. Taken together, we reveal the functional regulation of Q protein by tae-miR172 and transcriptional co-repressor TaTPL in controlling the bread wheat spike architecture. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Discovery of a novel oocyte-specific Krüppel-associated box domain-containing zinc finger protein required for early embryogenesis in cattle.

PubMed

Hand, Jacqelyn M; Zhang, Kun; Wang, Lei; Koganti, Prasanthi P; Mastrantoni, Kristen; Rajput, Sandeep K; Ashry, Mohamed; Smith, George W; Yao, Jianbo

2017-04-01

Zinc finger (ZNF) transcription factors interact with DNA through zinc finger motifs and play important roles in a variety of cellular functions including cell growth, proliferation, development, apoptosis, and intracellular signal transduction. One-third of ZNF proteins in metazoans contain a highly conserved N-terminal motif known as the Krüppel-associated box (KRAB) domain, which acts as a potent, DNA-binding dependent transcriptional repression module. Analysis of RNA-Seq data generated from a bovine oocyte cDNA library identified a novel transcript, which encodes a KRAB-containing ZNF transcription factor (named ZNFO). Characterization of ZNFO mRNA expression revealed that it is exclusively expressed in bovine oocytes and early embryos. A GFP reporter assay demonstrated that ZNFO protein localizes specifically to the nucleus, supporting its role in transcriptional regulation. To test the role of ZNFO in early embryonic development, zygotes were generated by in vitro maturation and fertilization of oocytes, and injected with small interfering RNA (siRNA) designed to knockdown ZNFO. Cleavage rates were not affected by ZNFO siRNA injection. However, embryonic development to 8- to 16-cell stage and blastocyst stage was significantly reduced relative to the uninjected and negative control siRNA-injected embryos. Further, interaction of ZNFO with the highly conserved co-factor, KRAB-associated protein-1 (KAP1), was demonstrated, and evidence supporting transcriptional repression by ZNFO was demonstrated using a GAL4-luciferase reporter system. Results of described studies demonstrate that ZNFO is a maternally-derived oocyte-specific nuclear factor required for early embryonic development in cattle, presumably functioning by repressing transcription. Copyright © 2017 Elsevier B.V. All rights reserved.

Highly repressible expression system for cloning genes that specify potentially toxic proteins.

PubMed Central

O'Connor, C D; Timmis, K N

1987-01-01

A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational. Images PMID:2443481

Transforming Growth Factor β Suppresses Peroxisome Proliferator–Activated Receptor γ Expression via Both SMAD Binding and Novel TGF-β Inhibitory Elements

PubMed Central

Lakshmi, Sowmya P.; Reddy, Aravind T.; Reddy, Raju C.

2017-01-01

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other’s expression, and such PPARγ downregulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here we show that TGF-β induces association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive corepressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. PMID:28100650

Mxi1 is a repressor of the c-Myc promoter and reverses activation by USF.

PubMed

Lee, T C; Ziff, E B

1999-01-08

The basic region/helix-loop-helix/leucine zipper (B-HLH-LZ) oncoprotein c-Myc is abundant in proliferating cells and forms heterodimers with Max protein that bind to E-box sites in DNA and stimulate genes required for proliferation. A second B-HLH-LZ protein, Mxi1, is induced during terminal differentiation, and forms heterodimers with Max that also bind E-boxes but tether the mSin3 transcriptional repressor protein along with histone deacetylase thereby antagonizing Myc-dependent activation. We show that Mxi1 also antagonizes Myc by a second pathway, repression of transcription from the major c-myc promoter, P2. Repression was independent of Mxi1 binding to mSin3 but dependent on the Mxi1 LZ and COOH-terminal sequences, including putative casein kinase II phosphorylation sites. Repression targeted elements of the myc P2 promoter core (-35/+10), where it reversed transactivation by the constitutive transcription factor, USF. We show that Zn2+ induction of a stably transfected, metallothionein promoter-regulated mxi1 gene blocked the ability of serum to induce transcription of the endogenous c-myc gene and cell entry into S phase. Thus, induction of Mxi1 in terminally differentiating cells may block Myc function by repressing the c-myc gene P2 promoter, as well as by antagonizing Myc-dependent transactivation through E-boxes.

DeoR repression at-a-distance only weakly responds to changes in interoperator separation and DNA topology.

PubMed Central

Dandanell, G

1992-01-01

The interoperator distance between a synthetic operator Os and the deoP2O2-galK fusion was varied between 46 and 176 bp. The repression of the deoP2 directed galK expression as a function of the interoperator distance (center-to-center) was measured in vivo in a single-copy system. The results show that the DeoR repressor efficiently can repress transcription at all the interoperator distances tested. The degree of repression depends very little on the spacing between the operators, however, a weak periodic dependency of 8-11 bp may exist. PMID:1437558

Repression of mesodermal fate by foxa, a key endoderm regulator of the sea urchin embryo.

PubMed

Oliveri, Paola; Walton, Katherine D; Davidson, Eric H; McClay, David R

2006-11-01

The foxa gene is an integral component of the endoderm specification subcircuit of the endomesoderm gene regulatory network in the Strongylocentrotus purpuratus embryo. Its transcripts become confined to veg2, then veg1 endodermal territories, and, following gastrulation, throughout the gut. It is also expressed in the stomodeal ectoderm. gatae and otx genes provide input into the pregastrular regulatory system of foxa, and Foxa represses its own transcription, resulting in an oscillatory temporal expression profile. Here, we report three separate essential functions of the foxa gene: it represses mesodermal fate in the veg2 endomesoderm; it is required in postgastrular development for the expression of gut-specific genes; and it is necessary for stomodaeum formation. If its expression is reduced by a morpholino, more endomesoderm cells become pigment and other mesenchymal cell types, less gut is specified, and the larva has no mouth. Experiments in which blastomere transplantation is combined with foxa MASO treatment demonstrate that, in the normal endoderm, a crucial role of Foxa is to repress gcm expression in response to a Notch signal, and hence to repress mesodermal fate. Chimeric recombination experiments in which veg2, veg1 or ectoderm cells contained foxa MASO show which region of foxa expression controls each of the three functions. These experiments show that the foxa gene is a component of three distinct embryonic gene regulatory networks.

Molecular mechanism underlying juvenile hormone-mediated repression of precocious larval-adult metamorphosis.

PubMed

Kayukawa, Takumi; Jouraku, Akiya; Ito, Yuka; Shinoda, Tetsuro

2017-01-31

Juvenile hormone (JH) represses precocious metamorphosis of larval to pupal and adult transitions in holometabolous insects. The early JH-inducible gene Krüppel homolog 1 (Kr-h1) plays a key role in the repression of metamorphosis as a mediator of JH action. Previous studies demonstrated that Kr-h1 inhibits precocious larval-pupal transition in immature larva via direct transcriptional repression of the pupal specifier Broad-Complex (BR-C). JH was recently reported to repress the adult specifier gene Ecdysone-induced protein 93F (E93); however, its mechanism of action remains unclear. Here, we found that JH suppressed ecdysone-inducible E93 expression in the epidermis of the silkworm Bombyx mori and in a B. mori cell line. Reporter assays in the cell line revealed that the JH-dependent suppression was mediated by Kr-h1. Genome-wide ChIP-seq analysis identified a consensus Kr-h1 binding site (KBS, 14 bp) located in the E93 promoter region, and EMSA confirmed that Kr-h1 directly binds to the KBS. Moreover, we identified a C-terminal conserved domain in Kr-h1 essential for the transcriptional repression of E93 Based on these results, we propose a mechanism in which JH-inducible Kr-h1 directly binds to the KBS site upstream of the E93 locus to repress its transcription in a cell-autonomous manner, thereby preventing larva from bypassing the pupal stage and progressing to precocious adult development. These findings help to elucidate the molecular mechanisms regulating the metamorphic genetic network, including the functional significance of Kr-h1, BR-C, and E93 in holometabolous insect metamorphosis.

Silencers, silencing, and heritable transcriptional states.

PubMed Central

Laurenson, P; Rine, J

1992-01-01

Three copies of the mating-type genes, which determine cell type, are found in the budding yeast Saccharomyces cerevisiae. The copy at the MAT locus is transcriptionally active, whereas identical copies of the mating-type genes at the HML and HMR loci are transcriptionally silent. Hence, HML and HMR, also known as the silent mating-type loci, are subject to a position effect. Regulatory sequences flank the silent mating-type loci and mediate repression of HML and HMR. These regulatory sequences are called silencers for their ability to repress the transcription of nearby genes in a distance- and orientation-independent fashion. In addition, a number of proteins, including the four SIR proteins, histone H4, and an alpha-acetyltransferase, are required for the complete repression of HML and HMR. Because alterations in the amino-terminal domain of histone H4 result in the derepression of the silent mating-type loci, the mechanism of repression may involve the assembly of a specific chromatin structure. A number of additional clues permit insight into the nature of repression at HML and HMR. First, an S phase event is required for the establishment of repression. Second, at least one gene appears to play a role in the establishment mechanism yet is not essential for the stable propagation of repression through many rounds of cell division. Third, certain aspects of repression are linked to aspects of replication. The silent mating-type loci share many similarities with heterochromatin. Furthermore, regions of S. cerevisiae chromosomes, such as telomeres, which are known to be heterochromatic in other organisms, require a subset of SIR proteins for repression. Further analysis of the transcriptional repression at the silent mating-type loci may lend insight into heritable repression in other eukaryotes. PMID:1480108

Translational Repression of NhaR, a Novel Pathway for Multi-Tier Regulation of Biofilm Circuitry by CsrA

PubMed Central

Pannuri, Archana; Yakhnin, Helen; Vakulskas, Christopher A.; Edwards, Adrianne N.; Babitzke, Paul

2012-01-01

The RNA binding protein CsrA (RsmA) represses biofilm formation in several proteobacterial species. In Escherichia coli, it represses the production of the polysaccharide adhesin poly-β-1,6-N-acetyl-d-glucosamine (PGA) by binding to the pgaABCD mRNA leader, inhibiting pgaA translation, and destabilizing this transcript. In addition, CsrA represses genes responsible for the synthesis of cyclic di-GMP, an activator of PGA production. Here we determined that CsrA also represses NhaR, a LysR-type transcriptional regulator which responds to elevated [Na+] and alkaline pH and activates the transcription of the pgaABCD operon. Gel shift studies revealed that CsrA binds at two sites in the 5′ untranslated segment of nhaR, one of which overlaps the Shine-Dalgarno sequence. An epitope-tagged NhaR protein, expressed from the nhaR chromosomal locus, and an nhaR posttranscriptional reporter fusion (PlacUV5-nhaR′-′lacZ) both showed robust repression by CsrA. Northern blotting revealed a complex transcription pattern for the nhaAR locus. Nevertheless, CsrA did not repress nhaR mRNA levels. Toeprinting assays showed that CsrA competes effectively with the ribosome for binding to the translation initiation region of nhaR. Together, these findings indicate that CsrA blocks nhaR translation. Epistasis studies with a pgaA-lacZ transcriptional fusion confirmed a model in which CsrA indirectly represses pgaABCD transcription via NhaR. We conclude that CsrA regulates the horizontally acquired pgaABCD operon and PGA biosynthesis at multiple levels. Furthermore, nhaR repression exemplifies an expanding role for CsrA as a global regulator of stress response systems. PMID:22037401

Targeted repression of AXIN2 and MYC gene expression using designer TALEs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S., E-mail: gsy3@psu.edu

Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacentmore » to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.« less

Mitosis-associated repression in development.

PubMed

Esposito, Emilia; Lim, Bomyi; Guessous, Ghita; Falahati, Hanieh; Levine, Michael

2016-07-01

Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail. © 2016 Esposito et al.; Published by Cold Spring Harbor Laboratory Press.

Coordinated Gene Regulation in the Initial Phase of Salt Stress Adaptation*

PubMed Central

Vanacloig-Pedros, Elena; Bets-Plasencia, Carolina; Pascual-Ahuir, Amparo; Proft, Markus

2015-01-01

Stress triggers complex transcriptional responses, which include both gene activation and repression. We used time-resolved reporter assays in living yeast cells to gain insights into the coordination of positive and negative control of gene expression upon salt stress. We found that the repression of “housekeeping” genes coincides with the transient activation of defense genes and that the timing of this expression pattern depends on the severity of the stress. Moreover, we identified mutants that caused an alteration in the kinetics of this transcriptional control. Loss of function of the vacuolar H+-ATPase (vma1) or a defect in the biosynthesis of the osmolyte glycerol (gpd1) caused a prolonged repression of housekeeping genes and a delay in gene activation at inducible loci. Both mutants have a defect in the relocation of RNA polymerase II complexes at stress defense genes. Accordingly salt-activated transcription is delayed and less efficient upon partially respiratory growth conditions in which glycerol production is significantly reduced. Furthermore, the loss of Hog1 MAP kinase function aggravates the loss of RNA polymerase II from housekeeping loci, which apparently do not accumulate at inducible genes. Additionally the Def1 RNA polymerase II degradation factor, but not a high pool of nuclear polymerase II complexes, is needed for efficient stress-induced gene activation. The data presented here indicate that the finely tuned transcriptional control upon salt stress is dependent on physiological functions of the cell, such as the intracellular ion balance, the protective accumulation of osmolyte molecules, and the RNA polymerase II turnover. PMID:25745106

Functional repression of PtSND2 represses growth and development by disturbing auxin biosynthesis, transport and signaling in transgenic poplar.

PubMed

Wang, Haihai; Tang, Renjie; Wang, Cuiting; Qi, Qi; Gai, Ying; Jiang, Xiangning; Zhang, Hongxia

2015-01-01

Using chimeric repressor silencing technology, we previously reported that functional repression of PtSND2 severely arrested wood formation in transgenic poplar (Populus). Here, we provide further evidence that auxin biosynthesis, transport and signaling were disturbed in these transgenic plants, leading to pleiotropic defects in their growth patterns, including inhibited leaf enlargement and vascular tissue development in the leaf central vein, suppressed cambial growth and fiber elongation in the stem, and arrested growth in the root system. Two transgenic lines, which displayed the most remarkable phenotypic deviation from the wild-type, were selected for detailed studies. In both transgenic lines, expression of genes for auxin biosynthesis, transport and signaling was down-regulated, and indole-3-acetic acid distribution was severely disturbed in the apical buds, leaves, stems and roots of field-grown transgenic plants. Transient transcription dual-luciferase assays of ProPtTYDC2::LUC, ProPttLAX2::LUC and ProPoptrIAA20.2::LUC in poplar protoplasts revealed that expression of auxin-related genes might be regulated by PtSND2 at the transcriptional level. All these results indicate that functional repression of PtSND2 altered auxin biosynthesis, transport and signaling, and thereby disturbed the normal growth and development of transgenic plants. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the Human T-Cell Leukemia Virus Type 1 Promoter▿

PubMed Central

Konesky, Kasey L.; Nyborg, Jennifer K.; Laybourn, Paul J.

2006-01-01

Upon infection of human T-cell leukemia virus type 1 (HTLV-1), the provirus is integrated into the host cell genome and subsequently packaged into chromatin that contains histone H1. Consequently, transcriptional activation of the virus requires overcoming the environment of chromatin and H1. To efficiently activate transcription, HTLV-1 requires the virally encoded protein Tax and cellular transcription factor CREB. Together Tax and CREB interact with three cis-acting promoter elements called viral cyclic-AMP response elements (vCREs). Binding of Tax and CREB to the vCREs promotes association of p300/CBP into the complex and leads to transcriptional activation. Therefore, to fully understand the mechanism of Tax transactivation, it is necessary to examine transcriptional activation from chromatin assembled with H1. Using a DNA template harboring the complete HTLV-1 promoter sequence and a highly defined recombinant assembly system, we demonstrate proper incorporation of histone H1 into chromatin. Addition of H1 to the chromatin template reduces HTLV-1 transcriptional activation through a novel mechanism. Specifically, H1 does not inhibit CREB or Tax binding to the vCREs or p300 recruitment to the promoter. Rather, H1 directly targets p300 acetyltransferase activity. Interestingly, in determining the mechanism of H1 repression, we have discovered a previously undefined function of Tax, overcoming the repressive effects of H1-chromatin. Tax specifically abrogates the H1 repression of p300 enzymatic activity in a manner independent of p300 recruitment and without displacement of H1 from the promoter. PMID:16943293

Repression of Virus-Induced Interferon A Promoters by Homeodomain Transcription Factor Ptx1

PubMed Central

Lopez, Sébastien; Island, Marie-Laure; Drouin, Jacques; Bandu, Marie-Thérese; Christeff, Nicolas; Darracq, Nicole; Barbey, Régine; Doly, Janine; Thomas, Dominique; Navarro, Sébastien

2000-01-01

Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE). The functional feature of the DNRE is to specifically act by repression of VRE-A activity. With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1). Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE. Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression. After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes. These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor. PMID:11003649

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TAF-4.

PubMed

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M; Lin, Rueyling

2008-10-03

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TFIID component TAF-4

PubMed Central

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M.; Lin, Rueyling

2008-01-01

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1–P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres, but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II following fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wildtype OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators. PMID:18854162

MTA family of coregulators in nuclear receptor biology and pathology

PubMed Central

Manavathi, Bramanandam; Singh, Kamini; Kumar, Rakesh

2007-01-01

Nuclear receptors (NRs) rely on coregulators (coactivators and corepressors) to modulate the transcription of target genes. By interacting with nucleosome remodeling complexes, NR coactivators potentiate transcription, whereas corepressors inhibit transcription of the target genes. Metastasis-associated proteins (MTA) represent an emerging family of novel NR coregulators. In general, MTA family members form independent nucleosome remodeling and deacetylation (NuRD) complexes and repress the transcription of different genes by recruiting histone deacetylases onto their target genes. However, MTA1 also acts as a coactivator in a promoter-context dependent manner. Recent findings that repression of estrogen receptor transactivation functions by MTA1, MTA1s, and MTA2 and regulation of MTA3 by estrogen signaling have indicated the significance of these proteins in NR signaling. Here, we highlight the action of MTA proteins on NR signaling and their roles in pathophysiological conditions. PMID:18174918

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Chunyan; Morohashi, Keita; Plotnikov, Alexander N.

Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes tri-methylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. Here in this study, we report discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show thatmore » a lead compound MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. Ultimately, these small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.« less

Adenovirus E1a prevents the retinoblastoma gene product from repressing the activity of a cellular transcription factor.

PubMed Central

Zamanian, M; La Thangue, N B

1992-01-01

The retinoblastoma (Rb) gene product forms a complex with the cellular transcription factor DRTF1, a property assumed to be important for mediating negative growth control because certain viral oncogenes, such as adenovirus E1a, prevent this interaction and mutant Rb alleles, which have lost the capacity to regulate growth, encode proteins that fail to associate with DRTF1. In this study, we show that the wild-type Rb protein can specifically repress transcription from promoters driven by DRTF1 whereas a naturally occurring mutant Rb protein cannot. Furthermore, Rb-mediated transcriptional repression can be overridden by adenovirus E1a; this requires regions in E1a necessary for cellular transformation. The Rb protein therefore acts in trans to repress the transcriptional activity of DRTF1 whereas adenovirus E1a prevents this interaction and thus maintains DRTF1 in a constitutively active state. The Rb protein and adenovirus E1a therefore have opposite effects on the activity of a common molecular target. Transcriptional repression mediated by the Rb protein and inactivation of repression by the E1a protein are likely to play an important role in mediating their biological effects. Images PMID:1385776

Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

PubMed Central

Johnson, Amanda N.; Weil, P. Anthony

2017-01-01

Repressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae. These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein- and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1AS). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways. PMID:28196871

Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.

PubMed

Zhang, Liyuan; Gu, Lingkun; Ringler, Patricia; Smith, Stanley; Rushton, Paul J; Shen, Qingxi J

2015-07-01

Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A BEN-domain-containing protein associates with heterochromatin and represses transcription.

PubMed

Sathyan, Kizhakke M; Shen, Zhen; Tripathi, Vidisha; Prasanth, Kannanganattu V; Prasanth, Supriya G

2011-09-15

In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression.

A BEN-domain-containing protein associates with heterochromatin and represses transcription

PubMed Central

Sathyan, Kizhakke M.; Shen, Zhen; Tripathi, Vidisha; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

2011-01-01

In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression. PMID:21914818

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase.

PubMed

Qu, Hongxia; Zheng, Liduan; Jiao, Wanju; Mei, Hong; Li, Dan; Song, Huajie; Fang, Erhu; Wang, Xiaojing; Li, Shiwang; Huang, Kai; Tong, Qiangsong

2016-09-06

Heparanase (HPSE) is the only endo-β-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression.

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

PubMed

Masuda, Junko; Kawamoto, Hiroshi; Strober, Warren; Takayama, Eiji; Mizutani, Akifumi; Murakami, Hiroshi; Ikawa, Tomokatsu; Kitani, Atsushi; Maeno, Narumi; Shigehiro, Tsukasa; Satoh, Ayano; Seno, Akimasa; Arun, Vaidyanath; Kasai, Tomonari; Fuss, Ivan J; Katsura, Yoshimoto; Seno, Masaharu

2016-12-01

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

PubMed

Gonzalez, Deyarina; Hamidi, Nurul; Del Sol, Ricardo; Benschop, Joris J; Nancy, Thomas; Li, Chao; Francis, Lewis; Tzouros, Manuel; Krijgsveld, Jeroen; Holstege, Frank C P; Conlan, R Steven

2014-02-18

Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

The molecular mechanism of SPOROCYTELESS/NOZZLE in controlling Arabidopsis ovule development

PubMed Central

Wei, Baoye; Zhang, Jinzhe; Pang, Changxu; Yu, Hao; Guo, Dongshu; Jiang, Hao; Ding, Mingxin; Chen, Zhuoyao; Tao, Qing; Gu, Hongya; Qu, Li-Jia; Qin, Genji

2015-01-01

Ovules are essential for plant reproduction and develop into seeds after fertilization. SPOROCYTELESS/NOZZLE (SPL/NZZ) has been known for more than 15 years as an essential factor for ovule development in Arabidopsis, but the biochemical nature of SPL function has remained unsolved. Here, we demonstrate that SPL functions as an adaptor-like transcriptional repressor. We show that SPL recruits TOPLESS/TOPLESS-RELATED (TPL/TPR) co-repressors to inhibit the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors. We reveal that SPL uses its EAR motif at the C-terminal end to recruit TPL/TPRs and its N-terminal part to bind and inhibit the TCPs. We demonstrate that either disruption of TPL/TPRs or overexpression of TCPs partially phenocopies the defects of megasporogenesis in spl. Moreover, disruption of TCPs causes phenotypes that resemble spl-D gain-of-function mutants. These results define the action mechanism for SPL, which along with TPL/TPRs controls ovule development by repressing the activities of key transcription factors. Our findings suggest that a similar gene repression strategy is employed by both plants and fungi to control sporogenesis. PMID:25378179

The antiphasic regulatory module comprising CDF5 and its antisense RNA FLORE links the circadian clock to photoperiodic flowering.

PubMed

Henriques, Rossana; Wang, Huan; Liu, Jun; Boix, Marc; Huang, Li-Fang; Chua, Nam-Hai

2017-11-01

Circadian rhythms of gene expression are generated by the combinatorial action of transcriptional and translational feedback loops as well as chromatin remodelling events. Recently, long noncoding RNAs (lncRNAs) that are natural antisense transcripts (NATs) to transcripts encoding central oscillator components were proposed as modulators of core clock function in mammals (Per) and fungi (frq/qrf). Although oscillating lncRNAs exist in plants, their functional characterization is at an initial stage. By screening an Arabidopsis thaliana lncRNA custom-made array we identified CDF5 LONG NONCODING RNA (FLORE), a circadian-regulated lncRNA that is a NAT of CDF5. Quantitative real-time RT-PCR confirmed the circadian regulation of FLORE, whereas GUS-staining and flowering time evaluation were used to determine its biological function. FLORE and CDF5 antiphasic expression reflects mutual inhibition in a similar way to frq/qrf. Moreover, whereas the CDF5 protein delays flowering by directly repressing FT transcription, FLORE promotes it by repressing several CDFs (CDF1, CDF3, CDF5) and increasing FT transcript levels, indicating both cis and trans function. We propose that the CDF5/FLORE NAT pair constitutes an additional circadian regulatory module with conserved (mutual inhibition) and unique (function in trans) features, able to fine-tune its own circadian oscillation, and consequently, adjust the onset of flowering to favourable environmental conditions. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Transforming growth factor β suppresses peroxisome proliferator-activated receptor γ expression via both SMAD binding and novel TGF-β inhibitory elements.

PubMed

Lakshmi, Sowmya P; Reddy, Aravind T; Reddy, Raju C

2017-04-24

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other's expression, and such PPARγ down-regulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here, we show that TGF-β induces the association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive co-repressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated the partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

The HTLV-1 Tax Oncoprotein Represses Ku80 Gene Expression

PubMed Central

Ducu, Razvan I.; Dayaram, Tajhal; Marriott, Susan J.

2011-01-01

The HTLV-I oncoprotein Tax interferes with DNA double strand break repair. Since non-homologous end joining (NHEJ) is a major pathway used to repair DNA double strand breaks we examined the effect of Tax on this pathway, with particular interest in the expression and function of Ku80, a critical component of the NHEJ pathway. Tax expression decreased Ku80 mRNA and protein levels, and repressed transcription from the Ku80 promoter. Conversely, Ku80 mRNA increased following siRNA knockdown of Tax in HTLV-I infected cells. Tax expression was associated with an elevated number of micronuclei and nucleoplasmic bridges, hallmarks of improper DNA double strand break repair. Our studies identified Tax as a transcriptional repressor of Ku80 that correlates with decreased DNA repair function. The reduction of Ku80 transcription by Tax may deplete the cell of an essential DNA break binding protein, resulting in reduced repair of DNA double strand breaks and accumulation genomic mutations. PMID:21571351

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 activation.

Stress-Responsive Mitogen-Activated Protein Kinases Interact with the EAR Motif of a Poplar Zinc Finger Protein and Mediate Its Degradation through the 26S Proteasome1[W][OA

PubMed Central

Hamel, Louis-Philippe; Benchabane, Meriem; Nicole, Marie-Claude; Major, Ian T.; Morency, Marie-Josée; Pelletier, Gervais; Beaudoin, Nathalie; Sheen, Jen; Séguin, Armand

2011-01-01

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors. PMID:21873571

Repression of Middle Sporulation Genes in Saccharomyces cerevisiae by the Sum1-Rfm1-Hst1 Complex Is Maintained by Set1 and H3K4 Methylation

PubMed Central

Jaiswal, Deepika; Jezek, Meagan; Quijote, Jeremiah; Lum, Joanna; Choi, Grace; Kulkarni, Rushmie; Park, DoHwan; Green, Erin M.

2017-01-01

The conserved yeast histone methyltransferase Set1 targets H3 lysine 4 (H3K4) for mono, di, and trimethylation and is linked to active transcription due to the euchromatic distribution of these methyl marks and the recruitment of Set1 during transcription. However, loss of Set1 results in increased expression of multiple classes of genes, including genes adjacent to telomeres and middle sporulation genes, which are repressed under normal growth conditions because they function in meiotic progression and spore formation. The mechanisms underlying Set1-mediated gene repression are varied, and still unclear in some cases, although repression has been linked to both direct and indirect action of Set1, associated with noncoding transcription, and is often dependent on the H3K4me2 mark. We show that Set1, and particularly the H3K4me2 mark, are implicated in repression of a subset of middle sporulation genes during vegetative growth. In the absence of Set1, there is loss of the DNA-binding transcriptional regulator Sum1 and the associated histone deacetylase Hst1 from chromatin in a locus-specific manner. This is linked to increased H4K5ac at these loci and aberrant middle gene expression. These data indicate that, in addition to DNA sequence, histone modification status also contributes to proper localization of Sum1. Our results also show that the role for Set1 in middle gene expression control diverges as cells receive signals to undergo meiosis. Overall, this work dissects an unexplored role for Set1 in gene-specific repression, and provides important insights into a new mechanism associated with the control of gene expression linked to meiotic differentiation. PMID:29066473

Very low amounts of glucose cause repression of the stress-responsive gene HSP12 in Saccharomyces cerevisiae.

PubMed

de Groot, E; Bebelman, J P; Mager, W H; Planta, R J

2000-02-01

Changing the growth mode of Saccharomyces cerevisiae by adding fermentable amounts of glucose to cells growing on a non-fermentable carbon source leads to rapid repression of general stress-responsive genes like HSP12. Remarkably, glucose repression of HSP12 appeared to occur even at very low glucose concentrations, down to 0.005%. Although these low levels of glucose do not induce fermentative growth, they do act as a growth signal, since upon addition of glucose to a concentration of 0.02%, growth rate increased and ribosomal protein gene transcription was up-regulated. In an attempt to elucidate how this type of glucose signalling may operate, several signalling mutants were examined. Consistent with the low amounts of glucose that elicit HSP12 repression, neither the main glucose-repression pathway nor cAMP-dependent activation of protein kinase A appeared to play a role in this regulation. Using mutants involved in glucose metabolism, evidence was obtained suggesting that glucose 6-phosphate serves as a signalling molecule. To identify the target for glucose repression on the promoter of the HSP12 gene, a promoter deletion series was used. The major transcription factors governing (stress-induced) transcriptional activation of HSP12 are Msn2p and Msn4p, binding to the general stress-responsive promoter elements (STREs). Surprisingly, glucose repression of HSP12 appeared to be independent of Msn2/4p: HSP12 transcription in glycerol-grown cells was unaffected in a deltamsn2deltamsn4 strain. Nevertheless, evidence was obtained that STRE-mediated transcription is the target of repression by low amounts of glucose. These data suggest that an as yet unidentified factor is involved in STRE-mediated transcriptional regulation of HSP12.

Transcriptional Regulation of JARID1B/KDM5B Histone Demethylase by Ikaros, Histone Deacetylase 1 (HDAC1), and Casein Kinase 2 (CK2) in B-cell Acute Lymphoblastic Leukemia*

PubMed Central

Wang, Haijun; Song, Chunhua; Ding, Yali; Pan, Xiaokang; Ge, Zheng; Tan, Bi-Hua; Gowda, Chandrika; Sachdev, Mansi; Muthusami, Sunil; Ouyang, Hongsheng; Lai, Liangxue; Francis, Olivia L.; Morris, Christopher L.; Abdel-Azim, Hisham; Dorsam, Glenn; Xiang, Meixian; Payne, Kimberly J.; Dovat, Sinisa

2016-01-01

Impaired function of the Ikaros (IKZF1) protein is associated with the development of high-risk B-cell precursor acute lymphoblastic leukemia (B-ALL). The mechanisms of Ikaros tumor suppressor activity in leukemia are unknown. Ikaros binds to the upstream regulatory elements of its target genes and regulates their transcription via chromatin remodeling. Here, we report that Ikaros represses transcription of the histone H3K4 demethylase, JARID1B (KDM5B). Transcriptional repression of JARID1B is associated with increased global levels of H3K4 trimethylation. Ikaros-mediated repression of JARID1B is dependent on the activity of the histone deacetylase, HDAC1, which binds to the upstream regulatory element of JARID1B in complex with Ikaros. In leukemia, JARID1B is overexpressed, and its inhibition results in cellular growth arrest. Ikaros-mediated repression of JARID1B in leukemia is impaired by pro-oncogenic casein kinase 2 (CK2). Inhibition of CK2 results in increased binding of the Ikaros-HDAC1 complex to the promoter of JARID1B, with increased formation of trimethylated histone H3 lysine 27 and decreased histone H3 Lys-9 acetylation. In cases of high-risk B-ALL that carry deletion of one Ikaros (IKZF1) allele, targeted inhibition of CK2 restores Ikaros binding to the JARID1B promoter and repression of JARID1B. In summary, the presented data suggest a mechanism through which Ikaros and HDAC1 regulate the epigenetic signature in leukemia: via regulation of JARID1B transcription. The presented data identify JARID1B as a novel therapeutic target in B-ALL and provide a rationale for the use of CK2 inhibitors in the treatment of high-risk B-ALL. PMID:26655717

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

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

PubMed

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

2009-05-08

Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1-3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target.

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

PubMed Central

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

2009-01-01

Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1–3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target. PMID:19244234

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.

Maternal Rest/Nrsf Regulates Zebrafish Behavior through snap25a/b

PubMed Central

Moravec, Cara E.; Samuel, John; Weng, Wei; Wood, Ian C.

2016-01-01

During embryonic development, regulation of gene expression is key to creating the many subtypes of cells that an organism needs throughout its lifetime. Recent work has shown that maternal genetics and environmental factors have lifelong consequences on diverse processes ranging from immune function to stress responses. The RE1-silencing transcription factor (Rest) is a transcriptional repressor that interacts with chromatin-modifying complexes to repress transcription of neural-specific genes during early development. Here we show that in zebrafish, maternally supplied rest regulates expression of target genes during larval development and has lifelong impacts on behavior. Larvae deprived of maternal rest are hyperactive and show atypical spatial preferences. Adult male fish deprived of maternal rest present with atypical spatial preferences in a novel environment assay. Transcriptome sequencing revealed 158 genes that are repressed by maternal rest in blastula stage embryos. Furthermore, we found that maternal rest is required for target gene repression until at least 6 dpf. Importantly, disruption of the RE1 sites in either snap25a or snap25b resulted in behaviors that recapitulate the hyperactivity phenotype caused by absence of maternal rest. Both maternal rest mutants and snap25a RE1 site mutants have altered primary motor neuron architecture that may account for the enhanced locomotor activity. These results demonstrate that maternal rest represses snap25a/b to modulate larval behavior and that early Rest activity has lifelong behavioral impacts. SIGNIFICANCE STATEMENT Maternal factors deposited in the oocyte have well-established roles during embryonic development. We show that, in zebrafish, maternal rest (RE1-silencing transcription factor) regulates expression of target genes during larval development and has lifelong impacts on behavior. The Rest transcriptional repressor interacts with chromatin-modifying complexes to limit transcription of neural genes. We identify several synaptic genes that are repressed by maternal Rest and demonstrate that snap25a/b are key targets of maternal rest that modulate larval locomotor activity. These results reveal that zygotic rest is unable to compensate for deficits in maternally supplied rest and uncovers novel temporal requirements for Rest activity, which has implications for the broad roles of Rest-mediated repression during neural development and in disease states. PMID:27605615

Disruption of histone modification and CARM1 recruitment by arsenic represses transcription at glucocorticoid receptor-regulated promoters.

PubMed

Barr, Fiona D; Krohmer, Lori J; Hamilton, Joshua W; Sheldon, Lynn A

2009-08-26

Chronic exposure to inorganic arsenic (iAs) found in the environment is one of the most significant and widespread environmental health risks in the U.S. and throughout the world. It is associated with a broad range of health effects from cancer to diabetes as well as reproductive and developmental anomalies. This diversity of diseases can also result from disruption of metabolic and other cellular processes regulated by steroid hormone receptors via aberrant transcriptional regulation. Significantly, exposure to iAs inhibits steroid hormone-mediated gene activation. iAs exposure is associated with disease, but is also used therapeutically to treat specific cancers complicating an understanding of iAs action. Transcriptional activation by steroid hormone receptors is accompanied by changes in histone and non-histone protein post-translational modification (PTM) that result from the enzymatic activity of coactivator and corepressor proteins such as GRIP1 and CARM1. This study addresses how iAs represses steroid receptor-regulated gene transcription. PTMs on histones H3 and H4 at the glucocorticoid receptor (GR)-activated mouse mammary tumor virus (MMTV) promoter were identified by chromatin immunoprecipitation analysis following exposure to steroid hormone+/-iAs. Histone H3K18 and H3R17 amino acid residues had significantly different patterns of PTMs after treatment with iAs. Promoter interaction of the coactivator CARM1 was disrupted, but the interaction of GRIP1, a p160 coactivator through which CARM1 interacts with a promoter, was intact. Over-expression of CARM1 was able to fully restore and GRIP1 partially restored iAs-repressed transcription indicating that these coactivators are functionally associated with iAs-mediated transcriptional repression. Both are essential for robust transcription at steroid hormone regulated genes and both are associated with disease when inappropriately expressed. We postulate that iAs effects on CARM1 and GRIP1 may underlie some of its therapeutic effects and as well be associated with its toxic effects.

SON and its alternatively spliced isoforms control MLL complex-mediated H3K4me3 and transcription of leukemia-associated genes

PubMed Central

Kim, Jung-Hyun; Baddoo, Melody C.; Park, Eun Young; Stone, Joshua K.; Park, Hyeonsoo; Butler, Thomas W.; Huang, Gang; Yan, Xiaomei; Pauli-Behn, Florencia; Myers, Richard M.; Tan, Ming; Flemington, Erik K.; Lim, Ssang-Taek; Erin Ahn, Eun-Young

2016-01-01

SUMMARY Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy, but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia. PMID:26990989

Untangling the Web: The Diverse Functions of the PIWI/piRNA Pathway

PubMed Central

Mani, Sneha Ramesh; Juliano, Celina E.

2014-01-01

SUMMARY Small RNAs impact several cellular processes through gene regulation. Argonaute proteins bind small RNAs to form effector complexes that control transcriptional and post-transcriptional gene expression. PIWI proteins belong to the Argonaute protein family, and bind PIWI-interacting RNAs (piRNAs). They are highly abundant in the germline, but are also expressed in some somatic tissues. The PIWI/piRNA pathway has a role in transposon repression in Drosophila, which occurs both by epigenetic regulation and post-transcriptional degradation of transposon mRNAs. These functions are conserved, but clear differences in the extent and mechanism of transposon repression exist between species. Mutations in piwi genes lead to the upregulation of transposon mRNAs. It is hypothesized that this increased transposon mobilization leads to genomic instability and thus sterility, although no causal link has been established between transposon upregulation and genome instability. An alternative scenario could be that piwi mutations directly affect genomic instability, and thus lead to increased transposon expression. We propose that the PIWI/piRNA pathway controls genome stability in several ways: suppression of transposons, direct regulation of chromatin architecture and regulation of genes that control important biological processes related to genome stability. The PIWI/piRNA pathway also regulates at least some, if not many, protein-coding genes, which further lends support to the idea that piwi genes may have broader functions beyond transposon repression. An intriguing possibility is that the PIWI/piRNA pathway is using transposon sequences to coordinate the expression of large groups of genes to regulate cellular function. PMID:23712694

Blimp-1 represses CD8 T cell expression of PD-1 using a feed-forward transcriptional circuit during acute viral infection

PubMed Central

Lu, Peiyuan; Youngblood, Benjamin A.; Austin, James W.; Rasheed Mohammed, Ata Ur; Butler, Royce; Ahmed, Rafi

2014-01-01

Programmed cell death 1 (PD-1) is an inhibitory immune receptor that regulates T cell function, yet the molecular events that control its expression are largely unknown. We show here that B lymphocyte–induced maturation protein 1 (Blimp-1)–deficient CD8 T cells fail to repress PD-1 during the early stages of CD8 T cell differentiation after acute infection with lymphocytic choriomeningitis virus (LCMV) strain Armstrong. Blimp-1 represses PD-1 through a feed-forward repressive circuit by regulating PD-1 directly and by repressing NFATc1 expression, an activator of PD-1 expression. Blimp-1 binding induces a repressive chromatin structure at the PD-1 locus, leading to the eviction of NFATc1 from its site. These data place Blimp-1 at an important phase of the CD8 T cell effector response and provide a molecular mechanism for its repression of PD-1. PMID:24590765

Recruitment of CREB1 and Histone Deacetylase 2 (HDAC2) to the Mouse Ltbp-1 Promoter Regulates its Constitutive Expression in a Dioxin Receptor-dependent Manner

PubMed Central

Gomez-Duran, Aurea; Ballestar, Esteban; Carvajal-Gonzalez, Jose M.; Marlowe, Jennifer L.; Puga, Alvaro; Esteller, Manel; Fernandez-Salguero, Pedro M.

2010-01-01

Latent TGFβ-binding protein 1 (LTBP-1) is a key regulator of TGFβ targeting and activation in the extracellular matrix. LTBP-1 is recognized as a major docking molecule to localize, and possibly to activate, TGFβ in the extracellular matrix. Despite this relevant function, the molecular mechanisms regulating Ltbp-1 transcription remain largely unknown. Previous results from our laboratory revealed that mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR) had increased Ltbp-1 mRNA expression and elevated TGFβ activity, suggesting that AhR repressed Ltbp-1 transcription. Here, we have cloned the mouse Ltbp-1 gene promoter and analysed its mechanism of transcriptional repression by AhR. Reporter gene assays, AhR over-expression and site-directed mutagenesis showed that basal Ltbp-1 transcription is AhR-dependent. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi) revealed that AhR regulates Ltbp-1 transcription by a mechanism involving recruitment of co-activators such as CREB1 and co-repressors such as HDAC2 to the Ltbp-1 promoter. In AhR-expressing (AhR+/+) MEF cells, the recruitment of HDAC1, 2 and 4 correlated with decreased K8H4 acetylation and impaired binding of pCREBSer133 to the Ltbp-1 promoter, likely maintaining a constitutive repressed state. AhR−/− MEF cells had the opposite pattern of HDACs and pCREB1Ser133 binding to Ltbp-1 promoter, and therefore, over-expressed Ltbp-1 mRNA. In agreement, siRNA for HDAC2 increased Ltbp-1 expression and K8H4 acetylation in AhR+/+ but not in AhR−/− MEF cells. We suggest that HDAC2 binding keeps Ltbp-1 promoter repressed in AhR+/+ MEF cells, whereas in AhR-null MEF cells the absence of HDAC2 and the binding of pCREBSer133 allow Ltbp-1 transcription. Thus, epigenetics can contribute to constitutive Ltbp-1 repression by a mechanism requiring AhR activity. PMID:18508077

Yeast carbon catabolite repression.

PubMed

Gancedo, J M

1998-06-01

Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated.

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

PubMed

Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

2015-05-01

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Mathematical Model of a Telomerase Transcriptional Regulatory Network Developed by Cell-Based Screening: Analysis of Inhibitor Effects and Telomerase Expression Mechanisms

PubMed Central

Bilsland, Alan E.; Stevenson, Katrina; Liu, Yu; Hoare, Stacey; Cairney, Claire J.; Roffey, Jon; Keith, W. Nicol

2014-01-01

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability. PMID:24550717

An evolutionarily conserved RNase-based mechanism for repression of transcriptional positive autoregulation

PubMed Central

Wurtmann, Elisabeth J.; Ratushny, Alexander V.; Pan, Min; Beer, Karlyn D.; Aitchison, John D.; Baliga, Nitin S.

2014-01-01

Summary It is known that environmental context influences the degree of regulation at the transcriptional and post-transcriptional levels. However, the principles governing the differential usage and interplay of regulation at these two levels are not clear. Here, we show that the integration of transcriptional and post-transcriptional regulatory mechanisms in a characteristic network motif drives efficient environment-dependent state transitions. Through phenotypic screening, systems analysis, and rigorous experimental validation, we discovered an RNase (VNG2099C) in Halobacterium salinarum that is transcriptionally co-regulated with genes of the aerobic physiologic state but acts on transcripts of the anaerobic state. Through modeling and experimentation we show that this arrangement generates an efficient state-transition switch, within which RNase-repression of a transcriptional positive autoregulation (RPAR) loop is critical for shutting down ATP-consuming active potassium uptake to reserve energy required for salinity adaptation under aerobic, high potassium, or dark conditions. Subsequently, we discovered that many Escherichia coli operons with energy-associated functions are also putatively controlled by RPAR indicating that this network motif may have evolved independently in phylogenetically distant organisms. Thus, our data suggest that interplay of transcriptional and post-transcriptional regulation in the RPAR motifis a generalized principle for efficient environment-dependent state transitions across prokaryotes. PMID:24612392

Arabidopsis MALE STERILITY1 Encodes a PHD-Type Transcription Factor and Regulates Pollen and Tapetum Development[W

PubMed Central

Ito, Takuya; Nagata, Noriko; Yoshiba, Yoshu; Ohme-Takagi, Masaru; Ma, Hong; Shinozaki, Kazuo

2007-01-01

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper–like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper–like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants. PMID:18032630

Msx1 Homeodomain Protein Represses the αGSU and GnRH Receptor Genes During Gonadotrope Development

PubMed Central

Xie, Huimin; Cherrington, Brian D.; Meadows, Jason D.; Witham, Emily A.

2013-01-01

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at −114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program. PMID:23371388

Msx1 homeodomain protein represses the αGSU and GnRH receptor genes during gonadotrope development.

PubMed

Xie, Huimin; Cherrington, Brian D; Meadows, Jason D; Witham, Emily A; Mellon, Pamela L

2013-03-01

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at -114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program.

Smad4 suppresses the tumorigenesis and aggressiveness of neuroblastoma through repressing the expression of heparanase

PubMed Central

Qu, Hongxia; Zheng, Liduan; Jiao, Wanju; Mei, Hong; Li, Dan; Song, Huajie; Fang, Erhu; Wang, Xiaojing; Li, Shiwang; Huang, Kai; Tong, Qiangsong

2016-01-01

Heparanase (HPSE) is the only endo-β-D-glucuronidase that is correlated with the progression of neuroblastoma (NB), the most common extracranial malignancy in childhood. However, the mechanisms underlying HPSE expression in NB still remain largely unknown. Herein, through analyzing cis-regulatory elements and mining public microarray datasets, we identified SMAD family member 4 (Smad4) as a crucial transcription regulator of HPSE in NB. We demonstrated that Smad4 repressed the HPSE expression at the transcriptional levels in NB cells. Mechanistically, Smad4 suppressed the HPSE expression through directly binding to its promoter and repressing the lymphoid enhancer binding factor 1 (LEF1)-facilitated transcription of HPSE via physical interaction. Gain- and loss-of-function studies demonstrated that Smad4 inhibited the growth, invasion, metastasis, and angiogenesis of NB cells in vitro and in vivo. Restoration of HPSE expression prevented the NB cells from changes in these biological features induced by Smad4. In clinical NB specimens, Smad4 was under-expressed and inversely correlated with HPSE levels, while LEF1 was highly expressed and positively correlated with HPSE expression. Patients with high Smad4 expression, low LEF1 or HPSE levels had greater survival probability. These results demonstrate that Smad4 suppresses the tumorigenesis and aggressiveness of NB through repressing the HPSE expression. PMID:27595937

T cell fates ‘zipped up’: how the Bach2 basic leucine zipper transcriptional repressor directs T cell differentiation and function1

PubMed Central

Richer, Martin J.; Lang, Mark L.; Butler, Noah S.

2016-01-01

Recent data illustrate a key role for the transcriptional regulator Bach2 in orchestrating T cell differentiation and function. Although Bach2 has a well-described role in B cell differentiation, emerging data show that Bach2 is a prototypical member of a novel class of transcription factors that regulates transcriptional activity in T cells at super enhancers, or regions of high transcriptional activity. Accumulating data demonstrate specific roles for Bach2 in favoring regulatory T cell generation, restraining effector T cell differentiation and potentiating memory T cell development. Evidence suggests that Bach2 regulates various facets of T cell function by repressing other key transcriptional regulator such as Blimp-1. This review examines our current understanding of the role of Bach2 in T cell function and highlights the growing evidence that this transcriptional repressor functions as a key regulator involved in maintenance of T cell quiescence, T cell subset differentiation and memory T cell generation. PMID:27496973

Small-Molecule Modulators of Methyl-Lysine Binding for the CBX7 Chromodomain

DOE PAGES

Ren, Chunyan; Morohashi, Keita; Plotnikov, Alexander N.; ...

2015-02-05

Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes tri-methylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. Here in this study, we report discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show thatmore » a lead compound MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. Ultimately, these small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.« less

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

PubMed

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

2008-11-28

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

The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae ▿

PubMed Central

Mallory, Michael J.; Law, Michael J.; Buckingham, Lela E.; Strich, Randy

2010-01-01

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains. PMID:20971827

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.

Sumoylation of the Basic Helix-Loop-Helix Transcription Factor Sharp-1 Regulates Recruitment of the Histone Methyltransferase G9a and Function in Myogenesis*

PubMed Central

Wang, Yaju; Shankar, Shilpa Rani; Kher, Devaki; Ling, Belinda Mei Tze; Taneja, Reshma

2013-01-01

Sumoylation is an important post-translational modification that alters the activity of many transcription factors. However, the mechanisms that link sumoylation to alterations in chromatin structure, which culminate in tissue specific gene expression, are not fully understood. In this study, we demonstrate that SUMO modification of the transcription factor Sharp-1 is required for its full transcriptional repression activity and function as an inhibitor of skeletal muscle differentiation. Sharp-1 is modified by sumoylation at two conserved lysine residues 240 and 255. Mutation of these SUMO acceptor sites in Sharp-1 does not impact its subcellular localization but attenuates its ability to act as a transcriptional repressor and inhibit myogenic differentiation. Consistently, co-expression of the SUMO protease SENP1 with wild type Sharp-1 abrogates Sharp-1-dependent inhibition of myogenesis. Interestingly, sumoylation acts as a signal for recruitment of the co-repressor G9a. Thus, enrichment of G9a, and histone H3 lysine 9 dimethylation (H3K9me2), a signature of G9a activity, is dramatically reduced at muscle promoters in cells expressing sumoylation-defective Sharp-1. Our findings demonstrate how sumoylation of Sharp-1 exerts an impact on chromatin structure and transcriptional repression of muscle gene expression through recruitment of G9a. PMID:23637228

A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock.

PubMed

Pruneda-Paz, Jose L; Breton, Ghislain; Para, Alessia; Kay, Steve A

2009-03-13

Transcriptional feedback loops constitute the molecular circuitry of the plant circadian clock. In Arabidopsis, a core loop is established between CCA1 and TOC1. Although CCA1 directly represses TOC1, the TOC1 protein has no DNA binding domains, which suggests that it cannot directly regulate CCA1. We established a functional genomic strategy that led to the identification of CHE, a TCP transcription factor that binds specifically to the CCA1 promoter. CHE is a clock component partially redundant with LHY in the repression of CCA1. The expression of CHE is regulated by CCA1, thus adding a CCA1/CHE feedback loop to the Arabidopsis circadian network. Because CHE and TOC1 interact, and CHE binds to the CCA1 promoter, a molecular linkage between TOC1 and CCA1 gene regulation is established.

Effects of cooperation between translating ribosome and RNA polymerase on termination efficiency of the Rho-independent terminator

PubMed Central

Li, Rui; Zhang, Qing; Li, Junbai; Shi, Hualin

2016-01-01

An experimental system was designed to measure in vivo termination efficiency (TE) of the Rho-independent terminator and position–function relations were quantified for the terminator tR2 in Escherichia coli. The terminator function was almost completely repressed when tR2 was located several base pairs downstream from the gene, and TE gradually increased to maximum values with the increasing distance between the gene and terminator. This TE–distance relation reflected a stochastic coupling of the ribosome and RNA polymerase (RNAP). Terminators located in the first 100 bp of the coding region can function efficiently. However, functional repression was observed when the terminator was located in the latter part of the coding region, and the degree of repression was determined by transcriptional and translational dynamics. These results may help to elucidate mechanisms of Rho-independent termination and reveal genomic locations of terminators and functions of the sequence that precedes terminators. These observations may have important applications in synthetic biology. PMID:26602687

An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites▿

PubMed Central

Seredick, Steve D.; Seredick, Barbara M.; Baker, David; Spiegelman, George B.

2009-01-01

In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect. PMID:19581368

Transcriptional repression mediated by repositioning of genes to the nuclear lamina.

PubMed

Reddy, K L; Zullo, J M; Bertolino, E; Singh, H

2008-03-13

Nuclear compartmentalization seems to have an important role in regulating metazoan genes. Although studies on immunoglobulin and other loci have shown a correlation between positioning at the nuclear lamina and gene repression, the functional consequences of this compartmentalization remain untested. We devised an approach for inducible tethering of genes to the inner nuclear membrane (INM), and tested the consequences of such repositioning on gene activity in mouse fibroblasts. Here, using three-dimensional DNA-immunoFISH, we demonstrate repositioning of chromosomal regions to the nuclear lamina that is dependent on breakdown and reformation of the nuclear envelope during mitosis. Moreover, tethering leads to the accumulation of lamin and INM proteins, but not to association with pericentromeric heterochromatin or nuclear pore complexes. Recruitment of genes to the INM can result in their transcriptional repression. Finally, we use targeted adenine methylation (DamID) to show that, as is the case for our model system, inactive immunoglobulin loci at the nuclear periphery are contacted by INM and lamina proteins. We propose that these molecular interactions may be used to compartmentalize and to limit the accessibility of immunoglobulin loci to transcription and recombination factors.

Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.

PubMed

Taniuchi, Ichiro; Osato, Motomi; Egawa, Takeshi; Sunshine, Mary Jean; Bae, Suk Chul; Komori, Toshihisa; Ito, Yoshiaki; Littman, Dan R

2002-11-27

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

Heterochromatin assembly and transcriptome repression by Set1 in coordination with a class II histone deacetylase

PubMed Central

Lorenz, David R; Meyer, Lauren F; Grady, Patrick J R; Meyer, Michelle M; Cam, Hugh P

2014-01-01

Histone modifiers play essential roles in controlling transcription and organizing eukaryotic genomes into functional domains. Here, we show that Set1, the catalytic subunit of the highly conserved Set1C/COMPASS complex responsible for histone H3K4 methylation (H3K4me), behaves as a repressor of the transcriptome largely independent of Set1C and H3K4me in the fission yeast Schizosaccharomyces pombe. Intriguingly, while Set1 is enriched at highly expressed and repressed loci, Set1 binding levels do not generally correlate with the levels of transcription. We show that Set1 is recruited by the ATF/CREB homolog Atf1 to heterochromatic loci and promoters of stress-response genes. Moreover, we demonstrate that Set1 coordinates with the class II histone deacetylase Clr3 in heterochromatin assembly at prominent chromosomal landmarks and repression of the transcriptome that includes Tf2 retrotransposons, noncoding RNAs, and regulators of development and stress-responses. Our study delineates a molecular framework for elucidating the functional links between transcriptome control and chromatin organization. DOI: http://dx.doi.org/10.7554/eLife.04506.001 PMID:25497836

Regulation of mRNA translation during mitosis.

PubMed

Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

2015-08-25

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

The Janus transcription factor HapX controls fungal adaptation to both iron starvation and iron excess

PubMed Central

Gsaller, Fabio; Hortschansky, Peter; Beattie, Sarah R; Klammer, Veronika; Tuppatsch, Katja; Lechner, Beatrix E; Rietzschel, Nicole; Werner, Ernst R; Vogan, Aaron A; Chung, Dawoon; Mühlenhoff, Ulrich; Kato, Masashi; Cramer, Robert A; Brakhage, Axel A; Haas, Hubertus

2014-01-01

Balance of physiological levels of iron is essential for every organism. In Aspergillus fumigatus and other fungal pathogens, the transcription factor HapX mediates adaptation to iron limitation and consequently virulence by repressing iron consumption and activating iron uptake. Here, we demonstrate that HapX is also essential for iron resistance via activating vacuolar iron storage. We identified HapX protein domains that are essential for HapX functions during either iron starvation or high-iron conditions. The evolutionary conservation of these domains indicates their wide-spread role in iron sensing. We further demonstrate that a HapX homodimer and the CCAAT-binding complex (CBC) cooperatively bind an evolutionary conserved DNA motif in a target promoter. The latter reveals the mode of discrimination between general CBC and specific HapX/CBC target genes. Collectively, our study uncovers a novel regulatory mechanism mediating both iron resistance and adaptation to iron starvation by the same transcription factor complex with activating and repressing functions depending on ambient iron availability. PMID:25092765

SUN2 Modulates HIV-1 Infection and Latency through Association with Lamin A/C To Maintain the Repressive Chromatin.

PubMed

Sun, Wei-Wei; Jiao, Shi; Sun, Li; Zhou, Zhaocai; Jin, Xia; Wang, Jian-Hua

2018-05-01

The postintegrational latency of HIV-1 is characterized by reversible silencing of long terminal repeat (LTR)-driven transcription of the HIV genome. It is known that the formation of repressive chromatin at the 5'-LTR of HIV-1 proviral DNA impedes viral transcription by blocking the recruitment of positive transcription factors. How the repressive chromatin is formed and modulated during HIV-1 infection remains elusive. Elucidation of which chromatin reassembly factor mediates the reorganization of chromatin is likely to facilitate the understanding of the host's modulation of HIV-1 transcription and latency. Here we revealed that "Sad1 and UNC84 domain containing 2" (SUN2), an inner nuclear membrane protein, maintained the repressive chromatin and inhibited HIV LTR-driven transcription of proviral DNA through an association with lamin A/C. Specifically, lamin A/C tethered SUN2 to the nucleosomes 1 and 2 of the HIV-1 5'-LTR to block the initiation and elongation of HIV-1 transcription. SUN2 knockdown converted chromatin to an active form and thus enhanced the phosphorylation of RNA polymerase II and its recruitment to the 5'-LTR HIV-1 proviral DNA, leading to reactivation of HIV-1 from latency. Conversely, the exogenous factors such as tumor necrosis factor alpha (TNF-α) induced reactivation, and the replication of HIV-1 led to the disassociation between SUN2 and lamin A/C, suggesting that disruption of the association between SUN2 and lamin A/C to convert the repressive chromatin to the active form might be a prerequisite for the initiation of HIV-1 transcription and replication. Together, our findings indicate that SUN2 is a novel chromatin reassembly factor that helps to maintain chromatin in a repressive state and consequently inhibits HIV-1 transcription. IMPORTANCE Despite the successful use of scores of antiretroviral drugs, HIV latency poses a major impediment to virus eradication. Elucidation of the mechanism of latency facilitates the discovery of new therapeutic strategies. It has been known that the formation of repressive chromatin at the 5'-LTR of HIV-1 proviral DNA impedes viral transcription and maintains viral latency, but how the repressive chromatin is formed and modulated during HIV-1 infection remains elusive. In this study, we performed in-depth virological and cell biological studies and discovered that an inner nuclear membrane protein, SUN2, is a novel chromatin reassembly factor that maintains repressive chromatin and thus modulates HIV-1 transcription and latency: therefore, targeting SUN2 may lead to new strategies for HIV cure. Copyright © 2018 Sun et al.

Yeast Carbon Catabolite Repression†

PubMed Central

Gancedo, Juana M.

1998-01-01

Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated. PMID:9618445

A Saccharomyces cerevisiae mitochondrial DNA fragment activates Reg1p-dependent glucose-repressible transcription in the nucleus.

PubMed

Santangelo, G M; Tornow, J

1997-12-01

As part of an effort to identify random carbon-source-regulated promoters in the Saccharomyces cerevisiae genome, we discovered that a mitochondrial DNA fragment is capable of directing glucose-repressible expression of a reporter gene. This fragment (CR24) originated from the mitochondrial genome adjacent to a transcription initiation site. Mutational analyses identified a GC cluster within the fragment that is required for transcriptional induction. Repression of nuclear CR24-driven transcription required Reg1p, indicating that this mitochondrially derived promoter is a member of a large group of glucose-repressible nuclear promoters that are similarly regulated by Reg1p. In vivo and in vitro binding assays indicated the presence of factors, located within the nucleus and the mitochondria, that bind to the GC cluster. One or more of these factors may provide a regulatory link between the nucleus and mitochondria.

Abasic pivot substitution harnesses target specificity of RNA interference

PubMed Central

Lee, Hye-Sook; Seok, Heeyoung; Lee, Dong Ha; Ham, Juyoung; Lee, Wooje; Youm, Emilia Moonkyung; Yoo, Jin Seon; Lee, Yong-Seung; Jang, Eun-Sook; Chi, Sung Wook

2015-01-01

Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA–target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼80–100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications. PMID:26679372

Cas9 gRNA engineering for genome editing, activation and repression

DOE PAGES

Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; ...

2015-09-07

Here we demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

The Mechanism of E2F/P130 Mediated Repression and Its Potential Tumor Suppressor Function in Breast Cancer

DTIC Science & Technology

2000-07-01

conserved PLDLS motif. JBC 273, 8549-8552 (1998). 4. Nibu, Y., Zhang, H., and Levine, M . Interaction of short-range repressors with Drosophila CtBP in...oncogenic transformation. Proc. Natl. Acad. Sci. USA 92, 10467-10471 (1995). 11. Poortinga,G., Watanabe, M . & Parkhurst,S.M. Drosophila CtBP: A hairy...Levine, M . Groucho and dCtBP mediate separate pathways of transcriptional repression in the Drosophila embryo. Developmental Biology 96, 535-540 (1999). 13

Dual Regulation of Bacillus subtilis kinB Gene Encoding a Sporulation Trigger by SinR through Transcription Repression and Positive Stringent Transcription Control.

PubMed

Fujita, Yasutaro; Ogura, Mitsuo; Nii, Satomi; Hirooka, Kazutake

2017-01-01

It is known that transcription of kinB encoding a trigger for Bacillus subtilis sporulation is under repression by SinR, a master repressor of biofilm formation, and under positive stringent transcription control depending on the adenine species at the transcription initiation nucleotide (nt). Deletion and base substitution analyses of the kinB promoter (P kinB ) region using lacZ fusions indicated that either a 5-nt deletion (Δ5, nt -61/-57, +1 is the transcription initiation nt) or the substitution of G at nt -45 with A (G-45A) relieved kinB repression. Thus, we found a pair of SinR-binding consensus sequences (GTTCTYT; Y is T or C) in an inverted orientation (SinR-1) between nt -57/-42, which is most likely a SinR-binding site for kinB repression. This relief from SinR repression likely requires SinI, an antagonist of SinR. Surprisingly, we found that SinR is essential for positive stringent transcription control of P kinB . Electrophoretic mobility shift assay (EMSA) analysis indicated that SinR bound not only to SinR-1 but also to SinR-2 (nt -29/-8) consisting of another pair of SinR consensus sequences in a tandem repeat arrangement; the two sequences partially overlap the '-35' and '-10' regions of P kinB . Introduction of base substitutions (T-27C C-26T) in the upstream consensus sequence of SinR-2 affected positive stringent transcription control of P kinB , suggesting that SinR binding to SinR-2 likely causes this positive control. EMSA also implied that RNA polymerase and SinR are possibly bound together to SinR-2 to form a transcription initiation complex for kinB transcription. Thus, it was suggested in this work that derepression of kinB from SinR repression by SinI induced by Spo0A∼P and occurrence of SinR-dependent positive stringent transcription control of kinB might induce effective sporulation cooperatively, implying an intimate interplay by stringent response, sporulation, and biofilm formation.

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

PubMed Central

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

2008-01-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komura, Jun-ichiro, E-mail: junkom@med.tohoku.ac.jp; Ikehata, Hironobu; Mori, Toshio

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cellsmore » was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.« less

JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuasa, Katsutoshi; Aoki, Natsumi; Hijikata, Takao, E-mail: hijikata@musashino-u.ac.jp

Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes.more » AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis. - Highlights: • JAZF1 promotes cell cycle progression and proliferation of myoblasts. • JAZF1 retards myogenic differentiation and hypertrophy of myotubes. • JAZF1 transcriptionally represses Mef2C and Mrf4 expression. • JAZF1 has an impact on the phosphorylation of AMPK.« less

The BET protein FSH functionally interacts with ASH1 to orchestrate global gene activity in Drosophila

PubMed Central

2013-01-01

Background The question of how cells re-establish gene expression states after cell division is still poorly understood. Genetic and molecular analyses have indicated that Trithorax group (TrxG) proteins are critical for the long-term maintenance of active gene expression states in many organisms. A generally accepted model suggests that TrxG proteins contribute to maintenance of transcription by protecting genes from inappropriate Polycomb group (PcG)-mediated silencing, instead of directly promoting transcription. Results and discussion Here we report a physical and functional interaction in Drosophila between two members of the TrxG, the histone methyltransferase ASH1 and the bromodomain and extraterminal family protein FSH. We investigated this interface at the genome level, uncovering a widespread co-localization of both proteins at promoters and PcG-bound intergenic elements. Our integrative analysis of chromatin maps and gene expression profiles revealed that the observed ASH1-FSH binding pattern at promoters is a hallmark of active genes. Inhibition of FSH-binding to chromatin resulted in global down-regulation of transcription. In addition, we found that genes displaying marks of robust PcG-mediated repression also have ASH1 and FSH bound to their promoters. Conclusions Our data strongly favor a global coactivator function of ASH1 and FSH during transcription, as opposed to the notion that TrxG proteins impede inappropriate PcG-mediated silencing, but are dispensable elsewhere. Instead, our results suggest that PcG repression needs to overcome the transcription-promoting function of ASH1 and FSH in order to silence genes. PMID:23442797

Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.

PubMed

Stennard, Fiona A; Costa, Mauro W; Lai, Donna; Biben, Christine; Furtado, Milena B; Solloway, Mark J; McCulley, David J; Leimena, Christiana; Preis, Jost I; Dunwoodie, Sally L; Elliott, David E; Prall, Owen W J; Black, Brian L; Fatkin, Diane; Harvey, Richard P

2005-05-01

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.

Scaffold attachment factor B suppresses HIV-1 infection of CD4+ cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat.

PubMed

Ma, Li; Sun, Li; Jin, Xia; Xiong, Si-Dong; Wang, Jian-Hua

2018-06-10

The 5' end of HIV-1 long terminal repeat (LTR) promoter plays an essential role in driving viral transcription and productive infection. Multiple host and viral factors regulate LTR activity and modulate HIV-1 latency. Manipulation of the HIV-1 LTR provides a potential therapeutic strategy for combating HIV-1 persistence. In this study, we identified an RNA-/DNA-binding protein, Scaffold Attachment Factor B (SAFB1) as a host-cell factor that represses HIV-1 transcription. We found that SAFB1 bound to HIV-1 5`-LTR and significantly repressed 5`-LTR-driven-viral transcription and HIV-1 infection of CD4 + T cells. Mechanistically, SAFB1-mediated repression of HIV-1 transcription and infection was independent of its RNA- and DNA-binding capacities, instead, by binding to phosphorylated RNA polymerase II (RNA pol II), SAFB1 blocked its recruitment to the HIV-1 LTR. Of note, the SAFB1-mediated repression of HIV-1 transcription from proviral DNA maintained HIV-1 latency in CD4 + T cells. In summary, our findings reveal that SAFB1 binds to HIV-1-LTR and physically interacts with phosphorylated RNA pol II, repressing HIV-1 transcription initiation and elongation. Our findings improve the understanding of host modulation of HIV-1 transcription and latency and provide a new host-cell target for improved anti-HIV-1 therapies. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Facilitated recycling protects human RNA polymerase III from repression by Maf1 in vitro.

PubMed

Cabart, Pavel; Lee, JaeHoon; Willis, Ian M

2008-12-26

Yeast cells synthesize approximately 3-6 million molecules of tRNA every cell cycle at a rate of approximately 2-4 transcripts/gene/s. This high rate of transcription is achieved through many rounds of reinitiation by RNA polymerase (pol) III on stable DNA-bound complexes of the initiation factor TFIIIB. Studies in yeast have shown that the rate of reinitiation is increased by facilitated recycling, a process that involves the repeated reloading of the polymerase on the same transcription unit. However, when nutrients become limiting or stress conditions are encountered, RNA pol III transcription is rapidly repressed through the action of the conserved Maf1 protein. Here we examine the relationship between Maf1-mediated repression and facilitated recycling in a human RNA pol III in vitro system. Using an immobilized template transcription assay, we demonstrate that facilitated recycling is conserved from yeast to humans. We assessed the ability of recombinant human Maf1 to inhibit different steps in transcription before and after preinitiation complex assembly. We show that recombinant Maf1 can inhibit the recruitment of TFIIIB and RNA pol III to immobilized templates. However, RNA pol III bound to preinitiation complexes or in elongation complexes is protected from repression by Maf1 and can undergo several rounds of initiation. This indicates that recombinant Maf1 is unable to inhibit facilitated recycling. The data suggest that additional biochemical steps may be necessary for rapid Maf1-dependent repression of RNA pol III transcription.

ABA Suppresses Root Hair Growth via the OBP4 Transcriptional Regulator1[OPEN

PubMed Central

Kawamura, Ayako; Schäfer, Sabine; Breuer, Christian; Shibata, Michitaro; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Matsui, Minami

2017-01-01

Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2. Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis. PMID:28167701

PtrWRKY19, a novel WRKY transcription factor, contributes to the regulation of pith secondary wall formation in Populus trichocarpa.

PubMed

Yang, Li; Zhao, Xin; Yang, Fan; Fan, Di; Jiang, Yuanzhong; Luo, Keming

2016-01-28

WRKY proteins are one of the largest transcription factor families in higher plants and play diverse roles in various biological processes. Previous studies have shown that some WRKY members act as negative regulators of secondary cell wall formation in pith parenchyma cells. However, the regulatory mechanism of pith secondary wall formation in tree species remains largely unknown. In this study, PtrWRKY19 encoding a homolog of Arabidopsis WRKY12 was isolated from Populus trichocarpa. PtrWRKY19 was expressed in all tissues tested, with highest expression in stems, especially in pith. PtrWRKY19 was located in the nucleus and functioned as a transcriptional repressor. Ectopic expression of PtrWRKY19 in an atwrky12 mutant successfully rescued the phenotype in pith cell walls caused by the defect of AtWRKY12, suggesting that PtrWRKY19 had conserved functions for homologous AtWRKY12. Overexpression of PtrWRKY19 in poplar plants led to a significant increase in the number of pith parenchyma cells. qRT-PCR analysis showed that lignin biosynthesis-related genes were repressed in transgenic plants. In transcient reporter assays, PtrWRKY19 was identified to repress transcription from the PtoC4H2 promoter containing the conserved W-box elements. These results indicated that PtrWRKY19 may function as a negative regulator of pith secondary wall formation in poplar.

Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis

PubMed Central

Nicolas, Pierre; Repoila, Francis; Bardowski, Jacek; Aymerich, Stéphane

2017-01-01

In eukaryotes, RNA species originating from pervasive transcription are regulators of various cellular processes, from the expression of individual genes to the control of cellular development and oncogenesis. In prokaryotes, the function of pervasive transcription and its output on cell physiology is still unknown. Most bacteria possess termination factor Rho, which represses pervasive, mostly antisense, transcription. Here, we investigate the biological significance of Rho-controlled transcription in the Gram-positive model bacterium Bacillus subtilis. Rho inactivation strongly affected gene expression in B. subtilis, as assessed by transcriptome and proteome analysis of a rho–null mutant during exponential growth in rich medium. Subsequent physiological analyses demonstrated that a considerable part of Rho-controlled transcription is connected to balanced regulation of three mutually exclusive differentiation programs: cell motility, biofilm formation, and sporulation. In the absence of Rho, several up-regulated sense and antisense transcripts affect key structural and regulatory elements of these differentiation programs, thereby suppressing motility and biofilm formation and stimulating sporulation. We dissected how Rho is involved in the activity of the cell fate decision-making network, centered on the master regulator Spo0A. We also revealed a novel regulatory mechanism of Spo0A activation through Rho-dependent intragenic transcription termination of the protein kinase kinB gene. Altogether, our findings indicate that distinct Rho-controlled transcripts are functional and constitute a previously unknown built-in module for the control of cell differentiation in B. subtilis. In a broader context, our results highlight the recruitment of the termination factor Rho, for which the conserved biological role is probably to repress pervasive transcription, in highly integrated, bacterium-specific, regulatory networks. PMID:28723971

Sumoylation of the net inhibitory domain (NID) is stimulated by PIAS1 and has a negative effect on the transcriptional activity of Net.

PubMed

Wasylyk, Christine; Criqui-Filipe, Paola; Wasylyk, Bohdan

2005-01-27

Net (Elk-3, Sap-2, Erp) and the related ternary complex factors Elk-1 and Sap-1 are effectors of multiple signalling pathways at the transcriptional level and play a key role in the dynamic regulation of gene expression. Net is distinct from Elk-1 and Sap-1, in that it is a strong repressor of transcription that is converted to an activator by the Ras/Erk signalling pathway. Two autonomous repression domains of Net, the NID and the CID, mediate repression. We have previously shown that the co-repressor CtBP is implicated in repression by the CID. In this report we show that repression by the NID involves a different pathway, sumoylation by Ubc9 and PIAS1. PIAS1 interacts with the NID in the two-hybrid assay and in vitro. Ubc9 and PIAS1 stimulate sumoylation in vivo of lysine 162 in the NID. Sumoylation of lysine 162 increases repression by Net and decreases the positive activity of Net. These results increase our understanding of how one of the ternary complex factors regulates transcription, and contribute to the understanding of how different domains of a transcription factor participate in the complexity of regulation of gene expression.

Carboxyl-Terminal Amino Acids 1052 to 1082 of the Latency-Associated Nuclear Antigen (LANA) Interact with RBP-Jκ and Are Responsible for LANA-Mediated RTA Repression

PubMed Central

Jin, Yi; He, Zhiheng; Liang, Deguang; Zhang, Quanzhi; Zhang, Hongxing; Deng, Qiang

2012-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is closely associated with several malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV can establish lifelong latency in the host, but the mechanism is not fully understood. Previous studies have proposed a feedback model in which the viral replication and transcription activator (RTA) can induce the expression of the latency-associated nuclear antigen (LANA) during early infection. LANA, in turn, represses transcription and RTA function to establish and maintain KSHV latency. The interaction between LANA and the recombination signal sequence binding protein Jκ (RBP-Jκ, also called CSL), a major transcriptional repressor of the Notch signaling pathway, is essential for RTA repression. In the present study, we show that the LANA carboxyl-terminal amino acids 1052 to 1082 are responsible for the LANA interaction with RBP-Jκ. The secondary structure of the LANA carboxyl terminus resembles the RBP-Jκ-associated module (RAM) of Notch receptor. Furthermore, deletion of the region of LANA residues 1052 to 1082 resulted in aberrant expression of RTA, leading to elevated viral lytic replication. For the first time, we dissected a conserved RBP-Jκ binding domain in LANA and demonstrated that this domain was indispensable for LANA-mediated repression of KSHV lytic genes, thus helping the virus maintain latency and control viral reactivation. PMID:22379075

Carboxyl-terminal amino acids 1052 to 1082 of the latency-associated nuclear antigen (LANA) interact with RBP-Jκ and are responsible for LANA-mediated RTA repression.

PubMed

Jin, Yi; He, Zhiheng; Liang, Deguang; Zhang, Quanzhi; Zhang, Hongxing; Deng, Qiang; Robertson, Erle S; Lan, Ke

2012-05-01

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is closely associated with several malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV can establish lifelong latency in the host, but the mechanism is not fully understood. Previous studies have proposed a feedback model in which the viral replication and transcription activator (RTA) can induce the expression of the latency-associated nuclear antigen (LANA) during early infection. LANA, in turn, represses transcription and RTA function to establish and maintain KSHV latency. The interaction between LANA and the recombination signal sequence binding protein Jκ (RBP-Jκ, also called CSL), a major transcriptional repressor of the Notch signaling pathway, is essential for RTA repression. In the present study, we show that the LANA carboxyl-terminal amino acids 1052 to 1082 are responsible for the LANA interaction with RBP-Jκ. The secondary structure of the LANA carboxyl terminus resembles the RBP-Jκ-associated module (RAM) of Notch receptor. Furthermore, deletion of the region of LANA residues 1052 to 1082 resulted in aberrant expression of RTA, leading to elevated viral lytic replication. For the first time, we dissected a conserved RBP-Jκ binding domain in LANA and demonstrated that this domain was indispensable for LANA-mediated repression of KSHV lytic genes, thus helping the virus maintain latency and control viral reactivation.

Molecular basis for repression of liver X receptor-mediated gene transcription by receptor-interacting protein 140

PubMed Central

Jakobsson, Tomas; Osman, Waffa; Gustafsson, Jan-Åke; Zilliacus, Johanna; Wärnmark, Anette

2007-01-01

Similarities in physiological roles of LXR (liver X receptors) and co-repressor RIP140 (receptor-interacting protein 140) in regulating energy homoeostasis and lipid and glucose metabolism suggest that the effects of LXR could at least partly be mediated by recruitment of the co-repressor RIP140. In the present study, we have elucidated the molecular basis for regulation of LXR transcriptional activity by RIP140. LXR is evenly localized in the nucleus and neither the N-terminal domain nor the LBD (ligand-binding domain) is necessary for nuclear localization. Both LXR subtypes, LXRα and LXRβ, interact with RIP140 and co-localize in diffuse large nuclear domains. Interaction and co-localization are dependent on the LBD of the receptor. The C-terminal domain of RIP140 is sufficient for full repressive effect. None of the C-terminal NR (nuclear receptor)-boxes is required for the co-repressor activity, whereas the NR-box-like motif as well as additional elements in the C-terminal region are required for full repressive function. The C-terminal NR-box-like motif is necessary for interaction with LXRβ, whereas additional elements are needed for strong interaction with LXRα. In conclusion, our results suggest that co-repression of LXR activity by RIP140 involves an atypical binding mode of RIP140 and a repression element in the RIP140 C-terminus. PMID:17391100

Mir-17-3p Controls Spinal Neural Progenitor Patterning by Regulating Olig2/Irx3 Cross-repressive Loop

PubMed Central

Chen, Jun-An; Huang, Yuan-Ping; Mazzoni, Esteban O.; Tan, G. Christopher; Zavadil, Jiri; Wichterle, Hynek

2011-01-01

SUMMARY Neural patterning relies on transcriptional cross-repressive interactions that ensure unequivocal assignment of neural progenitor identity to proliferating cells. Progenitors of spinal motor neurons (pMN) and V2 interneurons (p2) are specified by a pair of cross-repressive transcription factors Olig2 and Irx3. Lineage tracing revealed that many p2 progenitors transiently express the pMN marker Olig2 during spinal cord development. Here we demonstrate that the repression of Olig2 in p2 domain is controlled by mir-17-3p microRNA-mediated silencing of Olig2 mRNA. Mice lacking all microRNAs or just the mir-17~92 cluster manifest a dorsal shift in pMN/p2 boundary and impairment in the production of V2 interneurons. Our findings suggest that microRNA-mediated repression of Olig2 mRNA plays a critical role during the patterning of ventral spinal progenitor domains by shifting the balance of cross-repressive interactions between Olig2 and Irx3 transcription factors. PMID:21338882

Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells

PubMed Central

Miles, Wayne O; Korenjak, Michael; Griffiths, Lyra M; Dyer, Michael A; Provero, Paolo; Dyson, Nicholas J

2014-01-01

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE). PMID:25100735

Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells.

PubMed

Miles, Wayne O; Korenjak, Michael; Griffiths, Lyra M; Dyer, Michael A; Provero, Paolo; Dyson, Nicholas J

2014-10-01

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE). © 2014 The Authors.

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li

2013-02-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently ofmore » its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.« less

A WUSCHEL-Independent Stem Cell Specification Pathway Is Repressed by PHB, PHV and CNA in Arabidopsis.

PubMed

Lee, Chunghee; Clark, Steven E

2015-01-01

The homeostatic maintenance of stem cells that carry out continuous organogenesis at the shoot meristem is crucial for plant development. Key known factors act to signal between the stem cells and an underlying group of cells thought to act as the stem cell niche. In Arabidopsis thaliana the homeodomain transcription factor WUSCHEL (WUS) is essential for stem cell initiation and maintenance at shoot and flower meristems. Recent data suggest that the WUS protein may move from the niche cells directly into the stem cells to maintain stem cell identity. Here we provide evidence for a second, previously unknown, pathway for stem cell specification at shoot and flower meristems that bypasses the requirement for WUS. We demonstrate that this novel stem cell specification pathway is normally repressed by the activity of the HD-zip III transcription factors PHABULOSA (PHB), PHAVOLUTA (PHV) and CORONA (CNA). When de-repressed, this second stem cell pathway leads to an accumulation of stem cells and an enlargement of the stem cell niche. When de-repressed in a wus mutant background, this second stem cell pathway leads to functional meristems with largely normal cell layering and meristem morphology, activation of WUS cis regulatory elements, and extensive, but not indeterminate, organogenesis. Thus, WUS is largely dispensable for stem cell specification and meristem function, suggesting a set of key stem cell specification factors, competitively regulated by WUS and PHB/PHV/CNA, remain unidentified.

A WUSCHEL-Independent Stem Cell Specification Pathway Is Repressed by PHB, PHV and CNA in Arabidopsis

PubMed Central

Lee, Chunghee; Clark, Steven E.

2015-01-01

The homeostatic maintenance of stem cells that carry out continuous organogenesis at the shoot meristem is crucial for plant development. Key known factors act to signal between the stem cells and an underlying group of cells thought to act as the stem cell niche. In Arabidopsis thaliana the homeodomain transcription factor WUSCHEL (WUS) is essential for stem cell initiation and maintenance at shoot and flower meristems. Recent data suggest that the WUS protein may move from the niche cells directly into the stem cells to maintain stem cell identity. Here we provide evidence for a second, previously unknown, pathway for stem cell specification at shoot and flower meristems that bypasses the requirement for WUS. We demonstrate that this novel stem cell specification pathway is normally repressed by the activity of the HD-zip III transcription factors PHABULOSA (PHB), PHAVOLUTA (PHV) and CORONA (CNA). When de-repressed, this second stem cell pathway leads to an accumulation of stem cells and an enlargement of the stem cell niche. When de-repressed in a wus mutant background, this second stem cell pathway leads to functional meristems with largely normal cell layering and meristem morphology, activation of WUS cis regulatory elements, and extensive, but not indeterminate, organogenesis. Thus, WUS is largely dispensable for stem cell specification and meristem function, suggesting a set of key stem cell specification factors, competitively regulated by WUS and PHB/PHV/CNA, remain unidentified. PMID:26011610

Pumilio and nanos RNA-binding proteins counterbalance the transcriptional consequences of RB1 inactivation

PubMed Central

Miles, Wayne O; Dyson, Nicholas J

2014-01-01

The ability of the retinoblastoma protein (RB) tumor suppressor to repress transcription stimulated by the E2 promoter binding factors (E2F) is integral to its biological functions. Our recent report described a conserved feedback mechanism mediated by the RNA-binding proteins Pumilio and Nanos that increases in importance following RB loss and helps cells to tolerate deregulated E2F. PMID:27308363

Pumilio and nanos RNA-binding proteins counterbalance the transcriptional consequences of RB1 inactivation.

PubMed

Miles, Wayne O; Dyson, Nicholas J

2014-01-01

The ability of the retinoblastoma protein (RB) tumor suppressor to repress transcription stimulated by the E2 promoter binding factors (E2F) is integral to its biological functions. Our recent report described a conserved feedback mechanism mediated by the RNA-binding proteins Pumilio and Nanos that increases in importance following RB loss and helps cells to tolerate deregulated E2F.

Repression of enhancer II activity by a negative regulatory element in the hepatitis B virus genome.

PubMed Central

Lo, W Y; Ting, L P

1994-01-01

Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation-dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined effects of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression. Images PMID:8107237

Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

ABSTRACT Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeastYarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism inY. lipolytica. Deletion of the GATA transcription factor genesgzf3andgzf2resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells weremore » growing on a simple nitrogen source. Deletion ofgzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion ofgzf3results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, whilegzf2is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressormig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism. IMPORTANCENitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeastY. lipolyticato determine how the nitrogen source regulates lipid metabolism. We show that disruption of both activators and repressors of nitrogen catabolite repression leads to increased lipid accumulation via activation of carbon catabolite repression through an as yet uncharacterized method.« less

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.

Id2 Complexes with the SNAG Domain of Snai1 Inhibiting Snai1-Mediated Repression of Integrin β4

PubMed Central

Chang, Cheng; Yang, Xiaofang; Pursell, Bryan

2013-01-01

The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as α6β4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits β4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress β4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the β4 promoter and constrains the repressive function of Snai1. Disruption of the complex by depleting Id2 resulted in Snai1-mediated β4 repression with a concomitant increase in H3K27Me3 modification on the β4 promoter. These findings establish a novel function for Id2 in regulating Snai1 that has significant implications for the regulation of epithelial gene expression. PMID:23878399

Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa

PubMed Central

Wulf, Alexander; Campagne, Sébastien; Pei, Xue-Yuan; Forlani, Giada; Prindl, Konstantin; Abdou, Laetitia; Resch, Armin; Allain, Frederic H -T; Luisi, Ben F; Urlaub, Henning

2018-01-01

Abstract In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources. PMID:29244160

Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa.

PubMed

Sonnleitner, Elisabeth; Wulf, Alexander; Campagne, Sébastien; Pei, Xue-Yuan; Wolfinger, Michael T; Forlani, Giada; Prindl, Konstantin; Abdou, Laetitia; Resch, Armin; Allain, Frederic H-T; Luisi, Ben F; Urlaub, Henning; Bläsi, Udo

2018-02-16

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.

Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63

PubMed Central

Testoni, Barbara; Mantovani, Roberto

2006-01-01

p63 is a developmentally regulated transcription factor related to p53, which activates and represses specific genes. The human AEC (Ankyloblepharon–Ectodermal dysplasia-Clefting) and EEC (Ectrodactyly–Ectodermal dysplasia–Cleft lip/palate) syndromes are caused by missense mutations of p63, within the DNA-binding domain (EEC) or in the C-terminal sterile alpha motif domain (AEC). We show here that p63 represses transcription of cell-cycle G2/M genes by binding to multiple CCAAT core promoters in immortalized and primary keratinocytes. The CCAAT-activator NF-Y and ΔNp63α are associated in vivo and a conserved α-helix of the NF-YC histone fold is required. p63 AEC mutants, but not an EEC mutant, are incapable to bind NF-Y. ΔNp63α, but not the AEC mutants repress CCAAT-dependent transcription of G2/M genes. Chromatin immunoprecipitation recruitment assays establish that the AEC mutants are not recruited to G2/M promoters, while normally present on 14-3-3σ, which contains a sequence-specific binding site. Surprisingly, the EEC C306R mutant activates transcription. Upon keratinocytes differentiation, NF-Y and p63 remain bound to G2/M promoters, while HDACs are recruited, histones deacetylated, Pol II displaced and transcription repressed. Our data indicate that NF-Y is a molecular target of p63 and that inhibition of growth activating genes upon differentiation is compromised by AEC missense mutations. PMID:16473849

Nucleolar Association and Transcriptional Inhibition through 5S rDNA in Mammals

PubMed Central

Fedoriw, Andrew M.; Starmer, Joshua; Yee, Della; Magnuson, Terry

2012-01-01

Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent cis- elements, for such associations remains largely unknown. Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals. PMID:22275877

Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

PubMed

Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

2015-02-01

Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

MYB89 Transcription Factor Represses Seed Oil Accumulation1[OPEN

PubMed Central

Li, Dong; Jin, Changyu; Duan, Shaowei; Zhu, Yana; Qi, Shuanghui; Liu, Kaige; Gao, Chenhao; Ma, Haoli; Liao, Yuncheng

2017-01-01

In many higher plants, seed oil accumulation is precisely controlled by intricate multilevel regulatory networks, among which transcriptional regulation mainly influences oil biosynthesis. In Arabidopsis (Arabidopsis thaliana), the master positive transcription factors, WRINKLED1 (WRI1) and LEAFY COTYLEDON1-LIKE (L1L), are important for seed oil accumulation. We found that an R2R3-MYB transcription factor, MYB89, was expressed predominantly in developing seeds during maturation. Oil and major fatty acid biosynthesis in seeds was significantly promoted by myb89-1 mutation and MYB89 knockdown; thus, MYB89 was an important repressor during seed oil accumulation. RNA sequencing revealed remarkable up-regulation of numerous genes involved in seed oil accumulation in myb89 seeds at 12 d after pollination. Posttranslational activation of a MYB89-glucocorticoid receptor fusion protein and chromatin immunoprecipitation assays demonstrated that MYB89 inhibited seed oil accumulation by directly repressing WRI1 and five key genes and by indirectly suppressing L1L and 11 key genes involved in oil biosynthesis during seed maturation. These results help us to understand the novel function of MYB89 and provide new insights into the regulatory network of transcriptional factors controlling seed oil accumulation in Arabidopsis. PMID:27932421

Stochastic model for gene transcription on Drosophila melanogaster embryos

NASA Astrophysics Data System (ADS)

Prata, Guilherme N.; Hornos, José Eduardo M.; Ramos, Alexandre F.

2016-02-01

We examine immunostaining experimental data for the formation of stripe 2 of even-skipped (eve) transcripts on D. melanogaster embryos. An estimate of the factor converting immunofluorescence intensity units into molecular numbers is given. The analysis of the eve dynamics at the region of stripe 2 suggests that the promoter site of the gene has two distinct regimes: an earlier phase when it is predominantly activated until a critical time when it becomes mainly repressed. That suggests proposing a stochastic binary model for gene transcription on D. melanogaster embryos. Our model has two random variables: the transcripts number and the state of the source of mRNAs given as active or repressed. We are able to reproduce available experimental data for the average number of transcripts. An analysis of the random fluctuations on the number of eves and their consequences on the spatial precision of stripe 2 is presented. We show that the position of the anterior or posterior borders fluctuate around their average position by ˜1 % of the embryo length, which is similar to what is found experimentally. The fitting of data by such a simple model suggests that it can be useful to understand the functions of randomness during developmental processes.

Effects of cooperation between translating ribosome and RNA polymerase on termination efficiency of the Rho-independent terminator.

PubMed

Li, Rui; Zhang, Qing; Li, Junbai; Shi, Hualin

2016-04-07

An experimental system was designed to measure in vivo termination efficiency (TE) of the Rho-independent terminator and position-function relations were quantified for the terminator tR2 in Escherichia coli The terminator function was almost completely repressed when tR2 was located several base pairs downstream from the gene, and TE gradually increased to maximum values with the increasing distance between the gene and terminator. This TE-distance relation reflected a stochastic coupling of the ribosome and RNA polymerase (RNAP). Terminators located in the first 100 bp of the coding region can function efficiently. However, functional repression was observed when the terminator was located in the latter part of the coding region, and the degree of repression was determined by transcriptional and translational dynamics. These results may help to elucidate mechanisms of Rho-independent termination and reveal genomic locations of terminators and functions of the sequence that precedes terminators. These observations may have important applications in synthetic biology. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose.

PubMed

Ammar, Ehab M; Wang, Xiaoyi; Rao, Christopher V

2018-01-12

Catabolite repression refers to the process where the metabolism of one sugar represses the genes involved in metabolizing another sugar. While glucose provides the canonical example, many other sugars are also known to induce catabolite repression. However, less is known about the mechanism for catabolite repression by these non-glucose sugars. In this work, we investigated the mechanism of catabolite repression in the bacterium Escherichia coli during growth on lactose, L-arabinose, and D-xylose. The metabolism of these sugars is regulated in a hierarchical manner, where lactose is the preferred sugar, followed by L-arabinose, and then D-xylose. Previously, the preferential utilization of L-arabinose over D-xylose was found to result from transcriptional crosstalk. However, others have proposed that cAMP governs the hierarchical regulation of many non-glucose sugars. We investigated whether lactose-induced repression of L-arabinose and D-xylose gene expression is due to transcriptional crosstalk or cAMP. Our results demonstrate that it is due to cAMP and not transcriptional crosstalk. In addition, we found that repression is reciprocal, where both L-arabinose and D-xylose also repress the lactose gene expression, albeit to a lesser extent and also through a mechanism involving cAMP. Collectively, the results further our understanding of metabolism during growth on multiple sugars.

Redundant CArG Box Cis-motif Activity Mediates SHATTERPROOF2 Transcriptional Regulation during Arabidopsis thaliana Gynoecium Development

PubMed Central

Sehra, Bhupinder; Franks, Robert G.

2017-01-01

In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve. PMID:29085379

Repression by Homeoprotein Pitx1 of Virus-Induced Interferon A Promoters Is Mediated by Physical Interaction and trans Repression of IRF3 and IRF7

PubMed Central

Island, Marie-Laure; Mesplede, Thibault; Darracq, Nicole; Bandu, Marie-Thérèse; Christeff, Nicolas; Djian, Philippe; Drouin, Jacques; Navarro, Sébastien

2002-01-01

Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by the specific transcription activators IFN regulatory factor 3 (IRF3) and IRF-7 and the homeoprotein transcription repressor Pitx1. We now show that repression by Pitx1 does not appear to be due to the recruitment of histone deacetylases. On the other hand, Pitx1 inhibits the IRF3 and IRF7 transcriptional activity of the IFN-A11 and IFN-A5 promoters and interacts physically with IRF3 and IRF7. Pitx1 trans-repression activity maps to specific C-terminal domains, and the Pitx1 homeodomain is involved in physical interaction with IRF3 or IRF7. IRF3 is able to bind to the antisilencer region of the IFN-A4 promoter, which overrides the repressive activity of Pitx1. These results indicate that interaction between the Pitx1 homeodomain and IRF3 or IRF7 and the ability of the Pitx1 C-terminal repressor domains to block IFN-A11 and IFN-A5 but not IFN-A4 promoter activities may contribute to our understanding of the complex differential transcriptional activation, repression, and antirepression of the IFN-A genes. PMID:12242290

SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xia, Jun; Department of Respiratory Medicine, Jiangsu Provincial Hospital of Chinese Traditional Medicine; Wu, Xiaoyan

Highlights: Black-Right-Pointing-Pointer SIRT1 interacts with and deacetylates RFX5. Black-Right-Pointing-Pointer SIRT1 activation attenuates whereas SIRT1 inhibition enhances collagen repression by RFX5 in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 promotes cytoplasmic localization and proteasomal degradation of RFX5 and cripples promoter recruitment of RFX5. Black-Right-Pointing-Pointer IFN-{gamma} represses SIRT1 expression in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 agonist alleviates collagen repression by IFN-{gamma} in vascular smooth muscle cells. -- Abstract: Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanismmore » underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-{gamma} represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.« less

Chromatin Redistribution of the DEK Oncoprotein Represses hTERT Transcription in Leukemias12

PubMed Central

Karam, Maroun; Thenoz, Morgan; Capraro, Valérie; Robin, Jean-Philippe; Pinatel, Christiane; Lancon, Agnès; Galia, Perrine; Sibon, David; Thomas, Xavier; Ducastelle-Lepretre, Sophie; Nicolini, Franck; El-Hamri, Mohamed; Chelghoun, Youcef; Wattel, Eric; Mortreux, Franck

2014-01-01

Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis. PMID:24563617

AP-1 mediated transcriptional repression of matrix metalloproteinase-9 by recruitment of histone deacetylase 1 in response to interferon β.

PubMed

Mittelstadt, Megan L; Patel, Rekha C

2012-01-01

Matrix metalloproteinase-9 (MMP-9) is a 92 kDa zinc-dependant endopeptidase that degrades components of the extracellular matrix. Increased expression of MMP-9 is implicated in many pathological conditions including metastatic cancer, multiple sclerosis, and atherosclerosis. Although it has been widely noted that interferon-β (IFNβ) downregulates both the basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression at the transcriptional level, the molecular mechanism of this repression is poorly understood. In the present study we identify a novel mechanism for repression of MMP-9 transcription by IFNβ in HT1080 fibrosarcoma cells. Using reporter assays with promoter deletion constructs we show that IFNβ's inhibitory effects require a region of the promoter between -154 and -72, which contains an AP-1 binding site. Chromatin immunoprecipitation (ChIP) studies indicate that IFNβ increases histone deacetylase (HDAC)-1 recruitment to the MMP-9 promoter and reduces histone H3 acetylation, in addition to reduced NF-κB recruitment. ChIP analysis shows that IFNβ induced HDAC1 recruitment to the MMP-9 promoter and IFNβ mediated transcriptional repression is lost when the AP-1 binding site is inactivated by a point mutation. Altogether, our results establish that the repression of MMP-9 transcription in response to IFNβ occurs by the recruitment of HDAC1 via the proximal AP-1 binding site.

Evidence of the neuron-restrictive silencer factor (NRSF) interaction with Sp3 and its synergic repression to the mu opioid receptor (MOR) gene

PubMed Central

Kim, Chun Sung; Choi, Hack Sun; Hwang, Cheol Kyu; Song, Kyu Young; Lee, Byung-Kwon; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2006-01-01

Previously, we reported that the neuron-restrictive silencer element (NRSE) of mu opioid receptor (MOR) functions as a critical regulator to repress the MOR transcription in specific neuronal cells, depending on neuron-restriction silence factor (NRSF) expression levels [C.S.Kim, C.K.Hwang, H.S.Choi, K.Y.Song, P.Y.Law, L.N.Wei and H.H.Loh (2004) J. Biol. Chem., 279, 46464–46473]. Herein, we identify a conserved GC sequence next to NRSE region in the mouse MOR gene. The inhibition of Sp family factors binding to this GC box by mithramycin A led to a significant increase in the endogenous MOR transcription. In the co-immunoprecipitation experiment, NRSF interacted with the full-length Sp3 factor, but not with Sp1 or two short Sp3 isoforms. The sequence specific and functional binding by Sp3 at this GC box was confirmed by in vitro gel-shift assays using either in vitro translated proteins or nuclear extract, and by in vivo chromatin immunoprecipitation assays. Transient transfection assays showed that Sp3-binding site of the MOR gene is a functionally synergic repressor element with NRSE in NS20Y cells, but not in the NRSF negative PC12 cells. The results suggest that the synergic interaction between NRSF and Sp3 is required to negatively regulate MOR gene transcription and that transcription of MOR gene would be governed by the context of available transcription factors rather than by a master regulator. PMID:17130167

Regulation of mRNA translation during mitosis

PubMed Central

Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

2015-01-01

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ∼200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function. DOI: http://dx.doi.org/10.7554/eLife.07957.001 PMID:26305499

Disruption of DNA methylation-dependent long gene repression in Rett syndrome

PubMed Central

Gabel, Harrison W.; Kinde, Benyam Z.; Stroud, Hume; Gilbert, Caitlin S.; Harmin, David A.; Kastan, Nathaniel R.; Hemberg, Martin; Ebert, Daniel H.; Greenberg, Michael E.

2015-01-01

Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism1. MECP2 encodes a methyl-DNA-binding protein2 that has been proposed to function as a transcriptional repressor, but despite numerous studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 regulates transcription3–9. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain. PMID:25762136

Dimerization-induced corepressor binding and relaxed DNA-binding specificity are critical for PML/RARA-induced immortalization

PubMed Central

Zhou, Jun; Pérès, Laurent; Honoré, Nicole; Nasr, Rihab; Zhu, Jun; de Thé, Hugues

2006-01-01

The pathogenesis of acute promyelocytic leukemia involves the transcriptional repression of master genes of myeloid differentiation by the promyelocytic leukemia–retinoic acid receptor α (PML/RARA) oncogene. PML-enforced RARA homodimerization allows the tighter binding of corepressors, silencing RARA target genes. In addition, homodimerization dramatically extends the spectrum of DNA-binding sites of the fusion protein compared with those of normal RARA. Yet, any contribution of these two properties of PML/RARA to differentiation arrest and immortalization of primary mouse hematopoietic progenitors was unknown. We demonstrate that dimerization-induced silencing mediator of retinoid and thyroid receptors (SMRT)-enhanced binding and relaxed DNA-binding site specificity are both required for efficient immortalization. Thus, enforced RARA dimerization is critical not only for triggering transcriptional repression but also for extending the repertoire of target genes. Our studies exemplify how dimerization-induced gain of functions converts an unessential transcription factor into a dominant oncogenic protein. PMID:16757557

Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster.

PubMed

Tolhuis, Bas; de Wit, Elzo; Muijrers, Inhua; Teunissen, Hans; Talhout, Wendy; van Steensel, Bas; van Lohuizen, Maarten

2006-06-01

Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila melanogaster Kc cells. We found that Pc associates with large genomic regions of up to approximately 150 kb in size, hereafter referred to as 'Pc domains'. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains.

Repression by PRDM13 is critical for generating precision in neuronal identity

PubMed Central

Kollipara, Rahul K; Ma, Zhenzhong; Borromeo, Mark D; Chang, Joshua C

2017-01-01

The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development. PMID:28850031

DNA residence time is a regulatory factor of transcription repression

PubMed Central

Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji

2009-05-08

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependentmore » on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.« less

Epstein-Barr Virus (EBV) Latent Protein EBNA3A Directly Targets and Silences the STK39 Gene in B Cells Infected by EBV.

PubMed

Bazot, Quentin; Paschos, Kostas; Allday, Martin J

2018-04-01

Epstein-Barr virus (EBV) establishes latent infection in human B cells and is associated with a wide range of cancers. The EBV nuclear antigen 3 (EBNA3) family proteins are critical for B cell transformation and function as transcriptional regulators. It is well established that EBNA3A and EBNA3C cooperate in the regulation of cellular genes. Here, we demonstrate that the gene STK39 is repressed only by EBNA3A. This is the first example of a gene regulated only by EBNA3A in EBV-transformed lymphoblastoid cell lines (LCLs) without the help of EBNA3C. This was demonstrated using a variety of LCLs carrying either knockout, revertant, or conditional EBNA3 recombinants. Investigating the kinetics of EBNA3A-mediated changes in STK39 expression showed that STK39 becomes derepressed quickly after EBNA3A inactivation. This derepression is reversible as EBNA3A reactivation represses STK39 in the same cells expressing a conditional EBNA3A. STK39 is silenced shortly after primary B cell infection by EBV, and no STK39 -encoded protein (SPAK) is detected 3 weeks postinfection. Chromatin immunoprecipitation (ChIP) analysis indicates that EBNA3A directly binds to a regulatory region downstream of the STK39 transcription start site. For the first time, we demonstrated that the polycomb repressive complex 2 with the deposition of the repressive mark H3K27me3 is not only important for the maintenance of an EBNA3A target gene ( STK39 ) but is also essential for the initial establishment of its silencing. Finally, we showed that DNA methyltransferases are involved in the EBNA3A-mediated repression of STK39 IMPORTANCE EBV is well known for its ability to transform B lymphocytes to continuously proliferating lymphoblastoid cell lines. This is achieved in part by the reprogramming of cellular gene transcription by EBV transcription factors, including the EBNA3 proteins that play a crucial role in this process. In the present study, we found that EBNA3A epigenetically silences STK39 This is the first gene where EBNA3A has been found to exert its repressive role by itself, without needing its coregulators EBNA3B and EBNA3C. Furthermore, we demonstrated that the polycomb repressor complex is essential for EBNA3A-mediated repression of STK39 Findings in this study provide new insights into the regulation of cellular genes by the transcription factor EBNA3A. Copyright © 2018 Bazot et al.

PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy.

PubMed

Dong, Lijie; Nian, Hong; Shao, Yan; Zhang, Yan; Li, Qiutang; Yi, Yue; Tian, Fang; Li, Wenbo; Zhang, Hong; Zhang, Xiaomin; Wang, Fei; Li, Xiaorong

2015-05-01

Pathological retinal neovascularization, including retinopathy of prematurity and age-related macular degeneration, is the most common cause of blindness worldwide. Insulin-like growth factor-1 (IGF-1) has a direct mitogenic effect on endothelial cells, which is the basis of angiogenesis. Vascular endothelial growth factor (VEGF) activation in response to IGF-1 is well documented; however, the molecular mechanisms responsible for the termination of IGF-1 signaling are still not completely elucidated. Here, we show that the polypyrimidine tract-binding protein-associated splicing factor (PSF) is a potential negative regulator of VEGF expression induced by IGF stimulation. Functional analysis demonstrated that ectopic expression of PSF inhibits IGF-1-stimulated transcriptional activation and mRNA expression of the VEGF gene, whereas knockdown of PSF increased IGF-1-stimulated responses. PSF recruited Hakai to the VEGF transcription complex, resulting in inhibition of IGF-1-mediated transcription. Transfection with Hakai siRNA reversed the PSF-mediated transcriptional repression of VEGF gene transcription. In summary, these results show that PSF can repress the transcriptional activation of VEGF stimulated by IGF-1 via recruitment of the Hakai complex and delineate a novel regulatory mechanism of IGF-1/VEGF signaling that may have implications in the pathogenesis of neovascularization in ocular diseases.

mTORC1 Directly Phosphorylates and Regulates Human MAF1▿

PubMed Central

Michels, Annemieke A.; Robitaille, Aaron M.; Buczynski-Ruchonnet, Diane; Hodroj, Wassim; Reina, Jaime H.; Hall, Michael N.; Hernandez, Nouria

2010-01-01

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1. PMID:20516213

mTORC1 directly phosphorylates and regulates human MAF1.

PubMed

Michels, Annemieke A; Robitaille, Aaron M; Buczynski-Ruchonnet, Diane; Hodroj, Wassim; Reina, Jaime H; Hall, Michael N; Hernandez, Nouria

2010-08-01

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

PubMed Central

Kuchin, S; Yeghiayan, P; Carlson, M

1995-01-01

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter. Images Fig. 3 Fig. 4 Fig. 5 PMID:7732022

Competition between pre-mRNAs for the splicing machinery drives global regulation of splicing

PubMed Central

Munding, Elizabeth M.; Shiue, Lily; Katzman, Sol; Donohue, John Paul; Ares, Manuel

2013-01-01

Summary During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells RPG repression by rapamycin treatment also increases splicing efficiency. Down-regulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and pre-mRNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s) but also on those of competing pre-mRNAs. Competition between RNAs for limiting RNA processing factors appears to be a general condition in eukaryotic cells important for function of a variety of post-transcriptional control mechanisms including miRNA repression, polyadenylation and splicing. PMID:23891561

The transcription repressor, ZEB1, cooperates with CtBP2 and HDAC1 to suppress IL-2 gene activation in T cells.

PubMed

Wang, Jun; Lee, Seungsoo; Teh, Charis En-Yi; Bunting, Karen; Ma, Lina; Shannon, M Frances

2009-03-01

Activation of T cells leads to the induction of many cytokine genes that are required for appropriate immune responses, including IL-2, a key cytokine for T cell proliferation and homeostasis. The activating transcription factors such as nuclear factor of activated T cells, nuclear factor kappaB/Rel and activated protein-1 family members that regulate inducible IL-2 gene expression have been well documented. However, negative regulation of the IL-2 gene is less studied. Here we examine the role of zinc finger E-box-binding protein (ZEB) 1, a homeodomain/Zn finger transcription factor, as a repressor of IL-2 gene transcription. We show here that ZEB1 is expressed in non-stimulated and stimulated T cells and using chromatin immunoprecipitation assays we show that ZEB1 binds to the IL-2 promoter. Over-expression of ZEB1 can repress IL-2 promoter activity, as well as endogenous IL-2 mRNA production in EL-4 T cells, and this repression is dependent on the ZEB-binding site at -100. ZEB1 cooperates with the co-repressor C-terminal-binding protein (CtBP) 2 and with histone deacetylase 1 to repress the IL-2 promoter and this cooperation depends on the ZEB-binding site in the promoter as well as the Pro-X-Asp-Leu-Ser protein-protein interaction domain in CtBP2. Thus, ZEB1 may function to recruit a repressor complex to the IL-2 promoter.

The Transcription Factors TBX2 and TBX3 Interact with Human Papillomavirus 16 (HPV16) L2 and Repress the Long Control Region of HPVs

PubMed Central

Schneider, Marc A.; Scheffer, Konstanze D.; Bund, Timo; Boukhallouk, Fatima; Lambert, Carsten; Cotarelo, Cristina; Pflugfelder, Gert O.

2013-01-01

The minor capsid protein L2 of human papillomaviruses (HPVs) has multiple functions during the viral life cycle. Although L2 is required for effective invasion and morphogenesis, only a few cellular interaction partners are known so far. Using yeast two-hybrid screening, we identified the transcription factor TBX2 as a novel interaction partner of HPV type 16 (HPV16) L2. Coimmunoprecipitations and immunofluorescence analyses confirmed the L2-TBX2 interaction and revealed that L2 also interacts with TBX3, another member of the T-box family. Transcription of the early genes during HPV infection is under the control of an upstream enhancer and early promoter region, the long control region (LCR). In promoter-reporter gene assays, we observed that TBX2 and TBX3 repress transcription from the LCR and that this effect is enhanced by L2. Repression of the HPV LCR by TBX2/3 seems to be a conserved mechanism, as it was also observed with the LCRs of different HPV types. Finally, interaction of TBX2 with the LCR was detected by chromatin immunoprecipitation, and we found a strong colocalization of L2 and TBX2 in HPV16-positive cervical intraepithelial neoplasia (CIN) I-II tissue sections. These results suggest that TBX2/3 might play a role in the regulation of HPV gene expression during the viral life cycle. PMID:23388722

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation

PubMed Central

Nakagawa, Takeya; Kajitani, Takuya; Togo, Shinji; Masuko, Norio; Ohdan, Hideki; Hishikawa, Yoshitaka; Koji, Takehiko; Matsuyama, Toshifumi; Ikura, Tsuyoshi; Muramatsu, Masami; Ito, Takashi

2008-01-01

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation. PMID:18172164

Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs.

PubMed

Kutejova, Eva; Sasai, Noriaki; Shah, Ankita; Gouti, Mina; Briscoe, James

2016-03-21

In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The actin family protein ARP6 contributes to the structure and the function of the nucleolus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta

2015-08-21

The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed tomore » the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation.« less

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.

The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing.

PubMed

Mao, Y; Tamura, T; Yuki, Y; Abe, D; Tamada, Y; Imoto, S; Tanaka, H; Homma, H; Tagawa, K; Miyano, S; Okazawa, H

2016-04-28

In this study, we identify signaling network of necrotic cell death induced by transcriptional repression (TRIAD) by α-amanitin (AMA), the selective RNA polymerase II inhibitor, as a model of neurodegenerative cell death. We performed genetic screen of a knockdown (KD) fly library by measuring the ratio of transformation from pupa to larva (PL ratio) under TRIAD, and selected the cell death-promoting genes. Systems biology analysis of the positive genes mapped on protein-protein interaction databases predicted the signaling network of TRIAD and the core pathway including heterogeneous nuclear ribonucleoproteins (hnRNPs) and huntingtin (Htt). RNA sequencing revealed that AMA impaired transcription and RNA splicing of Htt, which is known as an endoplasmic reticulum (ER)-stabilizing molecule. The impairment in RNA splicing and PL ratio was rescued by overexpresion of hnRNP that had been also affected by transcriptional repression. Fly genetics with suppressor or expresser of Htt and hnRNP worsened or ameliorated the decreased PL ratio by AMA, respectively. Collectively, these results suggested involvement of RNA splicing and a regulatory role of the hnRNP-Htt axis in the process of the transcriptional repression-induced necrosis.

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting.

PubMed

Jackson, Angelyca A; Daniels, Emily F; Hammond, John H; Willger, Sven D; Hogan, Deborah A

2014-10-01

Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1%) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions. The Authors.

Repressive LTR Nucleosome Positioning by the BAF Complex Is Required for HIV Latency

PubMed Central

Hakre, Shweta; Moshkin, Yuri; Verdin, Eric; Mahmoudi, Tokameh

2011-01-01

Persistence of a reservoir of latently infected memory T cells provides a barrier to HIV eradication in treated patients. Several reports have implicated the involvement of SWI/SNF chromatin remodeling complexes in restricting early steps in HIV infection, in coupling the processes of integration and remodeling, and in promoter/LTR transcription activation and repression. However, the mechanism behind the seemingly contradictory involvement of SWI/SNF in the HIV life cycle remains unclear. Here we addressed the role of SWI/SNF in regulation of the latent HIV LTR before and after transcriptional activation. We determined the predicted nucleosome affinity of the LTR sequence and found a striking reverse correlation when compared to the strictly positioned in vivo LTR nucleosomal structure; sequences encompassing the DNase hypersensitive regions displayed the highest nucleosome affinity, while the strictly positioned nucleosomes displayed lower affinity for nucleosome formation. To examine the mechanism behind this reverse correlation, we used a combinatorial approach to determine DNA accessibility, histone occupancy, and the unique recruitment and requirement of BAF and PBAF, two functionally distinct subclasses of SWI/SNF at the LTR of HIV-infected cells before and after activation. We find that establishment and maintenance of HIV latency requires BAF, which removes a preferred nucleosome from DHS1 to position the repressive nucleosome-1 over energetically sub-optimal sequences. Depletion of BAF resulted in de-repression of HIV latency concomitant with a dramatic alteration in the LTR nucleosome profile as determined by high resolution MNase nucleosomal mapping. Upon activation, BAF was lost from the HIV promoter, while PBAF was selectively recruited by acetylated Tat to facilitate LTR transcription. Thus BAF and PBAF, recruited during different stages of the HIV life cycle, display opposing function on the HIV promoter. Our data point to the ATP-dependent BRG1 component of BAF as a putative therapeutic target to deplete the latent reservoir in patients. PMID:22140357

Repressive LTR nucleosome positioning by the BAF complex is required for HIV latency.

PubMed

Rafati, Haleh; Parra, Maribel; Hakre, Shweta; Moshkin, Yuri; Verdin, Eric; Mahmoudi, Tokameh

2011-11-01

Persistence of a reservoir of latently infected memory T cells provides a barrier to HIV eradication in treated patients. Several reports have implicated the involvement of SWI/SNF chromatin remodeling complexes in restricting early steps in HIV infection, in coupling the processes of integration and remodeling, and in promoter/LTR transcription activation and repression. However, the mechanism behind the seemingly contradictory involvement of SWI/SNF in the HIV life cycle remains unclear. Here we addressed the role of SWI/SNF in regulation of the latent HIV LTR before and after transcriptional activation. We determined the predicted nucleosome affinity of the LTR sequence and found a striking reverse correlation when compared to the strictly positioned in vivo LTR nucleosomal structure; sequences encompassing the DNase hypersensitive regions displayed the highest nucleosome affinity, while the strictly positioned nucleosomes displayed lower affinity for nucleosome formation. To examine the mechanism behind this reverse correlation, we used a combinatorial approach to determine DNA accessibility, histone occupancy, and the unique recruitment and requirement of BAF and PBAF, two functionally distinct subclasses of SWI/SNF at the LTR of HIV-infected cells before and after activation. We find that establishment and maintenance of HIV latency requires BAF, which removes a preferred nucleosome from DHS1 to position the repressive nucleosome-1 over energetically sub-optimal sequences. Depletion of BAF resulted in de-repression of HIV latency concomitant with a dramatic alteration in the LTR nucleosome profile as determined by high resolution MNase nucleosomal mapping. Upon activation, BAF was lost from the HIV promoter, while PBAF was selectively recruited by acetylated Tat to facilitate LTR transcription. Thus BAF and PBAF, recruited during different stages of the HIV life cycle, display opposing function on the HIV promoter. Our data point to the ATP-dependent BRG1 component of BAF as a putative therapeutic target to deplete the latent reservoir in patients.

Control of Transcriptional Repression of the Vitellogenin Receptor Gene in Largemouth Bass (Micropterus Salmoides) by Select Estrogen Receptors Isotypes

PubMed Central

Dominguez, Gustavo A.; Bisesi, Joseph H.; Kroll, Kevin J.; Denslow, Nancy D.; Sabo-Attwood, Tara

2014-01-01

The vitellogenin receptor (Vtgr) plays an important role in fish reproduction. This receptor functions to incorporate vitellogenin (Vtg), a macromolecule synthesized and released from the liver in the bloodstream, into oocytes where it is processed into yolk. Although studies have focused on the functional role of Vtgr in fish, the mechanistic control of this gene is still unexplored. Here we report the identification and analysis of the first piscine 5′ regulatory region of the vtgr gene which was cloned from largemouth bass (Micropterus salmoides). Using this putative promoter sequence, we investigated a role for hormones, including insulin and 17β-estradiol (E2), in transcriptional regulation through cell-based reporter assays. No effect of insulin was observed, however, E2 was able to repress transcriptional activity of the vtgr promoter through select estrogen receptor subtypes, Esr1 and Esr2a but not Esr2b. Electrophoretic mobility shift assay demonstrated that Esr1 likely interacts with the vtgr promoter region through half ERE and/or SP1 sites, in part. Finally we also show that ethinylestradiol (EE2), but not bisphenol-A (BPA), represses promoter activity similarly to E2. These results reveal for the first time that the Esr1 isoform may play an inhibitory role in the expression of LMB vtgr mRNA under the influence of E2, and potent estrogens such as EE2. In addition, this new evidence suggests that vtgr may be a target of select endocrine disrupting compounds through environmental exposures. PMID:25061109

Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements.

PubMed

Papantonis, Argyris; Sourmeli, Sissy; Lecanidou, Rena

2008-05-09

From the different cis-elements clustered on silkmoth chorion gene promoters, C/EBP binding sites predominate. Their sequence composition and dispersal vary amongst promoters of diverse developmental specificity. Occupancy of these sites by BmC/EBP was examined through Southwestern and ChIP assays modified to suit ovarian follicular cells. For the genes studied, binding of BmC/EBP coincided with the respective stages of transcriptional activation. However, the factor was reloaded on promoter sequences long after individual gene repression. Furthermore, suppression of BmC/EBP transcription in developing follicles resulted in de-regulation of chorion gene expression. A biphasic function of BmC/EBP, according to which it may act as both an activator and a repressor during silkmoth choriogenesis, is considered under the light of the presented data.

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.

Controlling transcription in human pluripotent stem cells using CRISPR-effectors.

PubMed

Genga, Ryan M; Kearns, Nicola A; Maehr, René

2016-05-15

The ability to manipulate transcription in human pluripotent stem cells (hPSCs) is fundamental for the discovery of key genes and mechanisms governing cellular state and differentiation. Recently developed CRISPR-effector systems provide a systematic approach to rapidly test gene function in mammalian cells, including hPSCs. In this review, we discuss recent advances in CRISPR-effector technologies that have been employed to control transcription through gene activation, gene repression, and epigenome engineering. We describe an application of CRISPR-effector mediated transcriptional regulation in hPSCs by targeting a synthetic promoter driving a GFP transgene, demonstrating the ease and effectiveness of CRISPR-effector mediated transcriptional regulation in hPSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Chromatin immunoprecipitation assays: application of ChIP-on-chip for defining dynamic transcriptional mechanisms in bone cells.

PubMed

van der Deen, Margaretha; Hassan, Mohammad Q; Pratap, Jitesh; Teplyuk, Nadiya M; Young, Daniel W; Javed, Amjad; Zaidi, Sayyed K; Lian, Jane B; Montecino, Martin; Stein, Janet L; Stein, Gary S; van Wijnen, Andre J

2008-01-01

Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as "ChIP-on-chip," has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.

c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sprowles, Amy; Robinson, Dan; Wu Yimi

2005-08-15

The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis tomore » define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli.« less

Altered Regulation of Escherichia coli Biotin Biosynthesis in BirA Superrepressor Mutant Strains

PubMed Central

Chakravartty, Vandana

2012-01-01

Transcription of the Escherichia coli biotin (bio) operon is directly regulated by the biotin protein ligase BirA, the enzyme that covalently attaches biotin to its cognate acceptor proteins. Binding of BirA to the bio operator requires dimerization of the protein, which is triggered by BirA-catalyzed synthesis of biotinoyl-adenylate (biotinoyl-5′-AMP), the obligatory intermediate of the ligation reaction. Although several aspects of this regulatory system are well understood, no BirA superrepressor mutant strains had been isolated. Such superrepressor BirA proteins would repress the biotin operon transcription in vivo at biotin concentrations well below those needed for repression by wild-type BirA. We isolated mutant strains having this phenotype by a combined selection-screening approach and resolved multiple mutations to give several birA superrepressor alleles, each having a single mutation, all of which showed repression dominant over that of the wild-type allele. All of these mutant strains repressed bio operon transcription in vivo at biotin concentrations that gave derepression of the wild-type strain and retained sufficient ligation activity for growth when overexpressed. All of the strains except that encoding G154D BirA showed derepression of bio operon transcription upon overproduction of a biotin-accepting protein. In BirA, G154D was a lethal mutation in single copy, and the purified protein was unable to transfer biotin from enzyme-bound biotinoyl-adenylate either to the natural acceptor protein or to a biotin-accepting peptide sequence. Consistent with the transcriptional repression data, each of the purified mutant proteins showed increased affinity for the biotin operator DNA in electrophoretic mobility shift assays. Surprisingly, although most of the mutations were located in the catalytic domain, all of those tested, except G154D BirA, had normal ligase activity. Most of the mutations that gave superrepressor phenotypes altered residues located close to the dimerization interface of BirA. However, two mutations were located at sites well removed from the interface. The properties of the superrepressor mutants strengthen and extend other data indicating that BirA function entails extensive interactions among the three domains of the protein and show that normal ligase activity does not ensure normal DNA binding. PMID:22210766

A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription

PubMed Central

2014-01-01

Background Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. Results To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. Conclusions This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription. PMID:24985467

A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription.

PubMed

Kuzmina, Alona; Verstraete, Nina; Galker, Sigal; Maatook, Maayan; Bensaude, Olivier; Taube, Ran

2014-07-01

Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription.

An upstream open reading frame represses expression of Lc, a member of the R/B family of maize transcriptional activators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Damiani, R.D. Jr.; Wessler, S.R.

1993-09-01

The R/B genes of maize encode a family of basic helix-loop-helix proteins that determine where and when the anthocyanin-pigment pathway will be expressed in the plant. Previous studies showed that allelic diversity among family members reflects differences in gene expression, specifically in transcription initiation. The authors present evidence that the R gene Lc is under translational control. They demonstrate that the 235-nt transcript leader of Lc represses expression 25- to 30-fold in an in vivo assay. Repression is mediated by the presence in cis of a 38-codon upstream open reading frame. Furthermore, the coding capacity of the upstream open readingmore » frame influences the magnitude of repression. It is proposed that translational control does not contribute to tissue specificity but prevents overexpression of the Lc protein. The diversity of promoter and 5' untranslated leader sequences among the R/B genes provides an opportunity to study the coevolution of transcriptional and translational mechanisms of gene regulation. 36 refs., 5 figs.« less

Nucleosome positioning in the regulatory region of SV40 chromatin correlates with the activation and repression of early and late transcription during infection

PubMed Central

Kumar, Meera Ajeet; Christensen, Kendra; Woods, Benjamin; Dettlaff, Ashley; Perley, Danielle; Scheidegger, Adam; Balakrishnan, Lata; Milavetz, Barry

2017-01-01

The location of nucleosomes in SV40 virions and minichromosomes isolated during infection were determined by next generation sequencing (NGS). The patterns of reads within the regulatory region of chromatin from wild-type virions indicated that micrococcal nuclease-resistant nucleosomes were specifically positioned at nt 5223 and nt 363, while in minichromosomes isolated 48 h post-infection we observed nuclease-resistant nucleosomes at nt 5119 and nt 212. The nucleosomes at nt 5223 and nt 363 in virion chromatin would be expected to repress early and late transcription, respectively. In virions from the mutant cs1085, which does not repress early transcription, we found that these two nucleosomes were significantly reduced compared to wild-type virions confirming a repressive role for them. In chromatin from cells infected for only 30 min with wild-type virus, we observed a significant reduction in the nucleosomes at nt 5223 and nt 363 indicating that the potential repression by these nucleosomes appeared to be relieved very early in infection. PMID:28126638

Regulation of growth-defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module

DOE Office of Scientific and Technical Information (OSTI.GOV)

Major, Ian T.; Yoshida, Yuki; Campos, Marcelo L.

The plant hormone jasmonate (JA) promotes the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins to relieve repression on diverse transcription factors (TFs) that execute JA responses. However, little is known about how combinatorial complexity among JAZ–TF interactions maintains control over myriad aspects of growth, development, reproduction, and immunity. We used loss-of-function mutations to define epistatic interactions within the core JA signaling pathway and to investigate the contribution of MYC TFs to JA responses in Arabidopsis thaliana. Constitutive JA signaling in a jaz quintuple mutant (jazQ) was largely eliminated by mutations that block JA synthesis or perception. Comparison of jazQ and amore » jazQ myc2 myc3 myc4 octuple mutant validated known functions of MYC2/3/4 in root growth, chlorophyll degradation,and susceptibility to the pathogen Pseudomonas syringae. We found that MYC TFs also control both the enhanced resistance of jazQ leaves to insect herbivory and restricted leaf growth of jazQ. Epistatic transcriptional profiles mirrored these phenotypes and further showed that triterpenoid biosynthetic and glucosinolate catabolic genes are up-regulated in jazQ independently of MYC TFs. Lastly, our study highlights the utility of genetic epistasis to unravel the complexities of JAZ–TF interactions and demonstrates that MYC TFs exert master control over a JAZ-repressible transcriptional hierarchy that governs growth–defense balance.« less

Regulation of growth-defense balance by the JASMONATE ZIM-DOMAIN (JAZ)-MYC transcriptional module

DOE PAGES

Major, Ian T.; Yoshida, Yuki; Campos, Marcelo L.; ...

2017-06-26

The plant hormone jasmonate (JA) promotes the degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins to relieve repression on diverse transcription factors (TFs) that execute JA responses. However, little is known about how combinatorial complexity among JAZ–TF interactions maintains control over myriad aspects of growth, development, reproduction, and immunity. We used loss-of-function mutations to define epistatic interactions within the core JA signaling pathway and to investigate the contribution of MYC TFs to JA responses in Arabidopsis thaliana. Constitutive JA signaling in a jaz quintuple mutant (jazQ) was largely eliminated by mutations that block JA synthesis or perception. Comparison of jazQ and amore » jazQ myc2 myc3 myc4 octuple mutant validated known functions of MYC2/3/4 in root growth, chlorophyll degradation,and susceptibility to the pathogen Pseudomonas syringae. We found that MYC TFs also control both the enhanced resistance of jazQ leaves to insect herbivory and restricted leaf growth of jazQ. Epistatic transcriptional profiles mirrored these phenotypes and further showed that triterpenoid biosynthetic and glucosinolate catabolic genes are up-regulated in jazQ independently of MYC TFs. Lastly, our study highlights the utility of genetic epistasis to unravel the complexities of JAZ–TF interactions and demonstrates that MYC TFs exert master control over a JAZ-repressible transcriptional hierarchy that governs growth–defense balance.« less

Superenhancer reprogramming drives a B-cell–epithelial transition and high-risk leukemia

PubMed Central

Hu, Yeguang; Zhang, Zhihong; Kashiwagi, Mariko; Yoshida, Toshimi; Joshi, Ila; Jena, Nilamani; Somasundaram, Rajesh; Emmanuel, Akinola Olumide; Sigvardsson, Mikael; Fitamant, Julien; El-Bardeesy, Nabeel; Gounari, Fotini; Van Etten, Richard A.; Georgopoulos, Katia

2016-01-01

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem–epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD–YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem–epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem–epithelial–B-cell phenotype that underlies high-risk B-ALL. PMID:27664237

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES).

PubMed

Han, Eun Hee; Gorman, Amanda A; Singh, Puja; Chi, Young-In

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that is impaired in the HNF1α mutation-driven diabetes. Copyright © 2015 Elsevier Inc. All rights reserved.

Glucose repression in Saccharomyces cerevisiae.

PubMed

Kayikci, Ömur; Nielsen, Jens

2015-09-01

Glucose is the primary source of energy for the budding yeast Saccharomyces cerevisiae. Although yeast cells can utilize a wide range of carbon sources, presence of glucose suppresses molecular activities involved in the use of alternate carbon sources as well as it represses respiration and gluconeogenesis. This dominant effect of glucose on yeast carbon metabolism is coordinated by several signaling and metabolic interactions that mainly regulate transcriptional activity but are also effective at post-transcriptional and post-translational levels. This review describes effects of glucose repression on yeast carbon metabolism with a focus on roles of the Snf3/Rgt2 glucose-sensing pathway and Snf1 signal transduction in establishment and relief of glucose repression. © FEMS 2015.

Single-cell analysis of Daxx and ATRX-dependent transcriptional repression

PubMed Central

Newhart, Alyshia; Rafalska-Metcalf, Ilona U.; Yang, Tian; Negorev, Dmitri G.; Janicki, Susan M.

2012-01-01

Summary Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function. PMID:22976303

Lineage specific expression of Polycomb Group Proteins in human embryonic stem cells in vitro.

PubMed

Pethe, Prasad; Pursani, Varsha; Bhartiya, Deepa

2015-05-01

Human embryonic (hES) stem cells are an excellent model to study lineage specification and differentiation into various cell types. Differentiation necessitates repression of specific genes not required for a particular lineage. Polycomb Group (PcG) proteins are key histone modifiers, whose primary function is gene repression. PcG proteins form complexes called Polycomb Repressive Complexes (PRCs), which catalyze histone modifications such as H2AK119ub1, H3K27me3, and H3K9me3. PcG proteins play a crucial role during differentiation of stem cells. The expression of PcG transcripts during differentiation of hES cells into endoderm, mesoderm, and ectoderm lineage is yet to be shown. In-house derived hES cell line KIND1 was differentiated into endoderm, mesoderm, and ectoderm lineages; followed by characterization using RT-PCR for HNF4A, CDX2, MEF2C, TBX5, SOX1, and MAP2. qRT-PCR and western blotting was performed to compare expression of PcG transcripts and proteins across all the three lineages. We observed that cells differentiated into endoderm showed upregulation of RING1B, BMI1, EZH2, and EED transcripts. Mesoderm differentiation was characterized by significant downregulation of all PcG transcripts during later stages. BMI1 and RING1B were upregulated while EZH2, SUZ12, and EED remained low during ectoderm differentiation. Western blotting also showed distinct expression of BMI1 and EZH2 during differentiation into three germ layers. Our study shows that hES cells differentiating into endoderm, mesoderm, and ectoderm lineages show distinct PcG expression profile at transcript and protein level. © 2015 International Federation for Cell Biology.

In silico modeling of epigenetic-induced changes in photoreceptor cis-regulatory elements.

PubMed

Hossain, Reafa A; Dunham, Nicholas R; Enke, Raymond A; Berndsen, Christopher E

2018-01-01

DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO , PDE6B, PAX6 , and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX-dependent transcription. Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

Antisense transcriptional interference mediates condition-specific gene repression in budding yeast.

PubMed

Nevers, Alicia; Doyen, Antonia; Malabat, Christophe; Néron, Bertrand; Kergrohen, Thomas; Jacquier, Alain; Badis, Gwenael

2018-05-18

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

Superrepression through Altered Corepressor-Activated Protein:Protein Interactions.

PubMed

He, Chenlu; Custer, Gregory; Wang, Jingheng; Matysiak, Silvina; Beckett, Dorothy

2018-02-20

Small molecules regulate transcription in both eukaryotes and prokaryotes by either enhancing or repressing assembly of transcription regulatory complexes. For allosteric transcription repressors, superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, because many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. Escherichia coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria, including corepressor binding, holorepressor dimerization, and binding of the dimer to DNA. A genetic screen has yielded superrepressor mutants that repress biotin operon transcription in vivo at biotin concentrations much lower than those required by the wild type repressor. In this work, isothermal titration calorimetry and sedimentation measurements were used to determine the superrepressor biotin binding and homodimerization properties. The results indicate that, although all variants exhibit biotin binding affinities similar to that measured for BirA wt , five of the six superrepressors show altered homodimerization energetics. Molecular dynamics simulations suggest that the altered dimerization results from perturbation of an electrostatic network that contributes to allosteric activation of BirA for dimerization. Modeling of the multistep repression complex assembly for these proteins reveals that the altered sensitivity of the transcription response to biotin concentration is readily explained solely by the altered superrepressor homodimerization energetics. These results highlight how coupled equilibria enable alterations in a transcription regulatory response to input signal through an indirect mechanism.

The Basic Helix-Loop-Helix Transcription Factor MYC2 Directly Represses PLETHORA Expression during Jasmonate-Mediated Modulation of the Root Stem Cell Niche in Arabidopsis[W][OA

PubMed Central

Chen, Qian; Sun, Jiaqiang; Zhai, Qingzhe; Zhou, Wenkun; Qi, Linlin; Xu, Li; Wang, Bao; Chen, Rong; Jiang, Hongling; Qi, Jing; Li, Xugang; Palme, Klaus; Li, Chuanyou

2011-01-01

The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin. PMID:21954460

Wnt-Mediated Repression via Bipartite DNA Recognition by TCF in the Drosophila Hematopoietic System

PubMed Central

Zhang, Chen U.; Blauwkamp, Timothy A.; Burby, Peter E.; Cadigan, Ken M.

2014-01-01

The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA. PMID:25144371

Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription.

PubMed Central

Russo, M W; Sevetson, B R; Milbrandt, J

1995-01-01

NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes which are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. In previous work, a structure/function analysis of NGFI-A revealed a 34-aa inhibitory domain that was hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7624335

A conserved regulatory mechanism in bifunctional biotin protein ligases.

PubMed

Wang, Jingheng; Beckett, Dorothy

2017-08-01

Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms. © 2017 The Protein Society.

Regulation of nitrogen metabolism by GATA zinc finger transcription factors in Yarrowia lipolytica

DOE PAGES

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.; ...

2017-02-15

Here, fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greatermore » accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.« less

Regulation of nitrogen metabolism by GATA zinc finger transcription factors in Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

Here, fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greatermore » accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.« less

PAX5 tyrosine phosphorylation by SYK co-operatively functions with its serine phosphorylation to cancel the PAX5-dependent repression of BLIMP1: A mechanism for antigen-triggered plasma cell differentiation.

PubMed

Inagaki, Yuichiro; Hayakawa, Fumihiko; Hirano, Daiki; Kojima, Yuki; Morishita, Takanobu; Yasuda, Takahiko; Naoe, Tomoki; Kiyoi, Hitoshi

2016-06-24

Plasma cell differentiation is initiated by antigen stimulation of the B cell receptor (BCR) and is regulated by BLIMP1. Prior to the stimulation of BCR, BLIMP1 is suppressed by PAX5, which is a key transcriptional repressor that maintains B cell identity. The upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed after the BCR stimulation. These events are considered to trigger plasma cell differentiation; however, the mechanisms responsible currently remain unclear. We herein demonstrated that the BCR signaling component, SYK, caused PAX5 tyrosine phosphorylation in vitro and in cells. Transcriptional repression on the BLIMP1 promoter by PAX5 was attenuated by this phosphorylation. The BCR stimulation induced the phosphorylation of SYK, tyrosine phosphorylation of PAX5, and up-regulation of BLIMP1 mRNA expression in B cells. The tyrosine phosphorylation of PAX5 co-operatively functioned with PAX5 serine phosphorylation by ERK1/2, which was our previous findings, to cancel the PAX5-dependent repression of BLIMP1. This co-operation may be a trigger for plasma cell differentiation. These results imply that PAX5 phosphorylation by a BCR signal is the initial event in plasma cell differentiation. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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

CIP, a cardiac Isl1-interacting protein, represses cardiomyocyte hypertrophy.

PubMed

Huang, Zhan-Peng; Young Seok, Hee; Zhou, Bin; Chen, Jinghai; Chen, Jian-Fu; Tao, Yazhong; Pu, William T; Wang, Da-Zhi

2012-03-16

Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well-established, the molecular events that inhibit or repress cardiac hypertrophy are less known. To identify and investigate novel regulators that modulate cardiac hypertrophy. Here, we report the identification, characterization, and functional examination of a novel cardiac Isl1-interacting protein (CIP). CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization, and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a cofactor of CIP. CIP directly interacted with Isl1, and we mapped the domains of these two proteins, which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the myocyte enhancer factor 2C. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Our studies therefore identify CIP as a novel regulator of cardiac hypertrophy.

Triggering Respirofermentative Metabolism in the Crabtree-Negative Yeast Pichia guilliermondii by Disrupting the CAT8 Gene

PubMed Central

Qi, Kai

2014-01-01

Pichia guilliermondii is a Crabtree-negative yeast that does not normally exhibit respirofermentative metabolism under aerobic conditions, and methods to trigger this metabolism may have applications for physiological study and industrial applications. In the present study, CAT8, which encodes a putative global transcriptional activator, was disrupted in P. guilliermondii. This yeast's ethanol titer increased by >20-fold compared to the wild type (WT) during aerobic fermentation using glucose. A comparative transcriptional analysis indicated that the expression of genes in the tricarboxylic acid cycle and respiratory chain was repressed in the CAT8-disrupted (ΔCAT8) strain, while the fermentative pathway genes were significantly upregulated. The respiratory activities in the ΔCAT8 strain, indicated by the specific oxygen uptake rate and respiratory state value, decreased to one-half and one-third of the WT values, respectively. In addition, the expression of HAP4, a transcriptional respiratory activator, was significantly repressed in the ΔCAT8 strain. Through disruption of HAP4, the ethanol production of P. guilliermondii was also increased, but the yield and titer were lower than that in the ΔCAT8 strain. A further transcriptional comparison between ΔCAT8 and ΔHAP4 strains suggested a more comprehensive reprogramming function of Cat8 in the central metabolic pathways. These results indicated the important role of CAT8 in regulating the glucose metabolism of P. guilliermondii and that the regulation was partially mediated by repressing HAP4. The strategy proposed here might be applicable to improve the aerobic fermentation capacity of other Crabtree-negative yeasts. PMID:24747899

Repression of YdaS Toxin Is Mediated by Transcriptional Repressor RacR in the Cryptic rac Prophage of Escherichia coli K-12.

PubMed

Krishnamurthi, Revathy; Ghosh, Swagatha; Khedkar, Supriya; Seshasayee, Aswin Sai Narain

2017-01-01

Horizontal gene transfer is a major driving force behind the genomic diversity seen in prokaryotes. The cryptic rac prophage in Escherichia coli K-12 carries the gene for a putative transcription factor RacR, whose deletion is lethal. We have shown that the essentiality of racR in E. coli K-12 is attributed to its role in transcriptionally repressing toxin gene(s) called ydaS and ydaT , which are adjacent to and coded divergently to racR . IMPORTANCE Transcription factors in the bacterium E. coli are rarely essential, and when they are essential, they are largely toxin-antitoxin systems. While studying transcription factors encoded in horizontally acquired regions in E. coli , we realized that the protein RacR, a putative transcription factor encoded by a gene on the rac prophage, is an essential protein. Here, using genetics, biochemistry, and bioinformatics, we show that its essentiality derives from its role as a transcriptional repressor of the ydaS and ydaT genes, whose products are toxic to the cell. Unlike type II toxin-antitoxin systems in which transcriptional regulation involves complexes of the toxin and antitoxin, repression by RacR is sufficient to keep ydaS transcriptionally silent.

Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein.

PubMed Central

Licht, J D; Hanna-Rose, W; Reddy, J C; English, M A; Ro, M; Grossel, M; Shaknovich, R; Hansen, U

1994-01-01

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region. Images PMID:8196644

VEGF ameliorates the ataxic phenotype in spinocerebellar ataxia type 1 (SCA1) mice

PubMed Central

Cvetanovic, Marija; Patel, Jay; Marti, Hugo H; Kini, Ameet R; Opal, Puneet

2012-01-01

SCA1 is an adult-onset, dominantly inherited neurodegenerative disease caused by expansion of a glutamine repeat tract in ATXN1. Although the precise function of ATXN1 remains elusive, it appears to play a role in transcriptional repression. We find that mutant ATXN1 suppresses transcription of the neurotrophic and angiogenic factor VEGF. We also show that genetic or pharmacologic replenishment of VEGF mitigates SCA1 pathogenesis, suggesting a novel therapeutic strategy for this incurable disease. PMID:22001907

Biophysical characterization of the basic cluster in the transcription repression domain of human MeCP2 with AT-rich DNA.

PubMed

Mushtaq, Ameeq Ul; Lee, Yejin; Hwang, Eunha; Bang, Jeong Kyu; Hong, Eunmi; Byun, Youngjoo; Song, Ji-Joon; Jeon, Young Ho

2018-01-01

MeCP2 is a chromatin associated protein which is highly expressed in brain and relevant with Rett syndrome (RTT). There are AT-hook motifs in MeCP2 which can bind with AT-rich DNA, suggesting a role in chromatin binding. Here, we report the identification and characterization of another AT-rich DNA binding motif (residues 295 to 313) from the C-terminal transcription repression domain of MeCP2 by nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC). This motif shows a micromolar affinity to AT-rich DNA, and it binds to the minor groove of DNA like AT-hook motifs. Together with the previous studies, our results provide an insight into a critical role of this motif in chromatin structure and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii.

PubMed

Yamasaki, Tomohito; Voshall, Adam; Kim, Eun-Jeong; Moriyama, Etsuko; Cerutti, Heriberto; Ohama, Takeshi

2013-12-01

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO▿ †

PubMed Central

Stratmann, Thomas; Madhusudan, S.; Schnetz, Karin

2008-01-01

The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ. PMID:18055596

Prolactin Signaling through the Short Form of Its Receptor Represses Forkhead Transcription Factor FOXO3 and Its Target Gene Galt Causing a Severe Ovarian Defect

PubMed Central

Halperin, Julia; Devi, Sangeeta Y.; Elizur, Shai; Stocco, Carlos; Shehu, Aurora; Rebourcet, Diane; Unterman, Terry G.; Leslie, Nancy D.; Le, Jamie; Binart, Nadine; Gibori, Geula

2008-01-01

Prolactin (PRL) is a hormone with over 300 biological activities. Although the signaling pathway downstream of the long form of its receptor (RL) has been well characterized, little is known about PRL actions upon activation of the short form (RS). Here, we show that mice expressing only RS exhibit an ovarian phenotype of accelerated follicular recruitment followed by massive follicular death leading to premature ovarian failure. Consequently, RS-expressing ovaries of young adults are depleted of functional follicles and formed mostly by interstitium. We also show that activation of RS represses the expression of the transcription factor Forkhead box O3 (FOXO3) and that of the enzyme galactose-1-phosphate uridyltransferase (Galt), two proteins known to be essential for normal follicular development. Our finding that FOXO3 regulates the expression of Galt and enhances its transcriptional activity indicates that it is the repression of FOXO3 by PRL acting through RS that prevents Galt expression in the ovary and causes follicular death. Coexpression of RL with RS prevents PRL inhibition of Galt, and the ovarian defect is no longer seen in RS transgenic mice that coexpress RL, suggesting that RL prevents RS-induced ovarian impairment. In summary, we show that PRL signals through RS and causes, in the absence of RL, a severe ovarian pathology by repressing the expression of FOXO3 and that of its target gene Galt. We also provide evidence of a link between the premature ovarian failure seen in mice expressing RS and in mice with FOXO3 gene deletion as well as in human with Galt mutation. PMID:17975019

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction.

PubMed

Bossi, Flavia; Fan, Jue; Xiao, Jun; Chandra, Lilyana; Shen, Max; Dorone, Yanniv; Wagner, Doris; Rhee, Seung Y

2017-06-26

The molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. To identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation. We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.

Nitrogen Metabolite Repression of Metabolism and Virulence in the Human Fungal Pathogen Cryptococcus neoformans

PubMed Central

Lee, I. Russel; Chow, Eve W. L.; Morrow, Carl A.; Djordjevic, Julianne T.; Fraser, James A.

2011-01-01

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine—three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°–40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection. PMID:21441208

Function and regulation of the Mediator complex.

PubMed

Conaway, Ronald C; Conaway, Joan Weliky

2011-04-01

Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected complexities in the way Mediator participates in signal transduction. Copyright © 2011 Elsevier Ltd. All rights reserved.

Epigenetic modification of histone 3 lysine 27: mediator subunit MED25 is required for the dissociation of polycomb repressive complex 2 from the promoter of cytochrome P450 2C9.

PubMed

Englert, Neal A; Luo, George; Goldstein, Joyce A; Surapureddi, Sailesh

2015-01-23

The Mediator complex is vital for the transcriptional regulation of eukaryotic genes. Mediator binds to nuclear receptors at target response elements and recruits chromatin-modifying enzymes and RNA polymerase II. Here, we examine the involvement of Mediator subunit MED25 in the epigenetic regulation of human cytochrome P450 2C9 (CYP2C9). MED25 is recruited to the CYP2C9 promoter through association with liver-enriched HNF4α, and we show that MED25 influences the H3K27 status of the HNF4α binding region. This region was enriched for the activating marker H3K27ac and histone acetyltransferase CREBBP after MED25 overexpression but was trimethylated when MED25 expression was silenced. The epigenetic regulator Polycomb repressive complex (PRC2), which represses expression by methylating H3K27, plays an important role in target gene regulation. Silencing MED25 correlated with increased association of PRC2 not only with the promoter region chromatin but with HNF4α itself. We confirmed the involvement of MED25 for fully functional preinitiation complex recruitment and transcriptional output in vitro. Formaldehyde-assisted isolation of regulatory elements (FAIRE) revealed chromatin conformation changes that were reliant on MED25, indicating that MED25 induced a permissive chromatin state that reflected increases in CYP2C9 mRNA. For the first time, we showed evidence that a functionally relevant human gene is transcriptionally regulated by HNF4α via MED25 and PRC2. CYP2C9 is important for the metabolism of many exogenous chemicals including pharmaceutical drugs as well as endogenous substrates. Thus, MED25 is important for regulating the epigenetic landscape resulting in transcriptional activation of a highly inducible gene, CYP2C9. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs

PubMed Central

El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

2015-01-01

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs. PMID:26162096

AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

PubMed

El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

2015-01-01

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock.

PubMed

Agrawal, Parul; Hardin, Paul E

2016-12-07

Circadian clocks in eukaryotes keep time via cell-autonomous transcriptional feedback loops. A well-characterized example of such a transcriptional feedback loop is in Drosophila, where CLOCK-CYCLE (CLK-CYC) complexes activate transcription of period (per) and timeless (tim) genes, rising levels of PER-TIM complexes feed-back to repress CLK-CYC activity, and degradation of PER and TIM permits the next cycle of CLK-CYC transcription. The timing of CLK-CYC activation and PER-TIM repression is regulated posttranslationally, in part through rhythmic phosphorylation of CLK, PER, and TIM. Previous behavioral screens identified several kinases that control CLK, PER, and TIM levels, subcellular localization, and/or activity, but two phosphatases that function within the clock were identified through the analysis of candidate genes from other pathways or model systems. To identify phosphatases that play a role in the clock, we screened clock cell-specific RNA interference (RNAi) knockdowns of all annotated protein phosphatases and protein phosphatase regulators in Drosophila for altered activity rhythms. This screen identified 19 protein phosphatases that lengthened or shortened the circadian period by ≥1 hr (p ≤ 0.05 compared to controls) or were arrhythmic. Additional RNAi lines, transposon inserts, overexpression, and loss-of-function mutants were tested to independently confirm these RNAi phenotypes. Based on genetic validation and molecular analysis, 15 viable protein phosphatases remain for future studies. These candidates are expected to reveal novel features of the circadian timekeeping mechanism in Drosophila that are likely to be conserved in all animals including humans. Copyright © 2016 Agrawal and Hardin.

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

PubMed Central

Foda, Bardees M.; Singh, Upinder

2015-01-01

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

Molecular architecture of polycomb repressive complexes

PubMed Central

Chittock, Emily C.; Latwiel, Sebastian; Miller, Thomas C.R.

2017-01-01

The polycomb group (PcG) proteins are a large and diverse family that epigenetically repress the transcription of key developmental genes. They form three broad groups of polycomb repressive complexes (PRCs) known as PRC1, PRC2 and Polycomb Repressive DeUBiquitinase, each of which modifies and/or remodels chromatin by distinct mechanisms that are tuned by having variable compositions of core and accessory subunits. Until recently, relatively little was known about how the various PcG proteins assemble to form the PRCs; however, studies by several groups have now allowed us to start piecing together the PcG puzzle. Here, we discuss some highlights of recent PcG structures and the insights they have given us into how these complexes regulate transcription through chromatin. PMID:28202673

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

PubMed

Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A; Ramaswamy, Suresh; Plant, Tony M; Ojeda, Sergio R

2015-12-16

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

Regulation of sugar transport and metabolism by the Candida albicans Rgt1 transcriptional repressor.

PubMed

Sexton, Jessica A; Brown, Victoria; Johnston, Mark

2007-10-01

The ability of the fungal pathogen Candida albicans to cause systemic infections depends in part on the function of Hgt4, a cell surface sugar sensor. The orthologues of Hgt4 in Saccharomyces cerevisiae, Snf3 and Rgt2, initiate a signalling cascade that inactivates Rgt1, a transcriptional repressor of genes encoding hexose transporters. To determine whether Hgt4 functions similarly through the C. albicans orthologue of Rgt1, we analysed Cargt1 deletion mutants. We found that Cargt1 mutants are sensitive to the glucose analogue 2-deoxyglucose, a phenotype probably due to uncontrolled expression of genes encoding glucose transporters. Indeed, transcriptional profiling revealed that expression of about two dozen genes, including multiple HGT genes encoding hexose transporters, is increased in the Cargt1 mutant in the absence of sugars, suggesting that CaRgt1 represses expression of several HGT genes under this condition. Some of the HGT genes (probably encoding high-affinity transporters) are also repressed by high levels of glucose, and we show that this repression is mediated by CaMig1, the orthologue of the major glucose-activated repressor in S. cerevisiae, but not by its paralogue CaMig2. Therefore, CaRgt1 and CaMig1 collaborate to control expression of C. albicans hexose transporters in response to different levels of sugars. We were surprised to find that CaRgt1 also regulates expression of GAL1, suggesting that regulation of galactose metabolism in C. albicans is unconventional. Finally, Cargt1 mutations cause cells to hyperfilament, and suppress the hypofilamented phenotype of an hgt4 mutant, indicating that the Hgt4 glucose sensor may affect filamentation by modulating sugar import and metabolism via CaRgt1. Copyright 2007 John Wiley & Sons, Ltd.

Control of transcriptional repression of the vitellogenin receptor gene in largemouth bass (Micropterus salmoides) by select estrogen receptors isotypes.

PubMed

Dominguez, Gustavo A; Bisesi, Joseph H; Kroll, Kevin J; Denslow, Nancy D; Sabo-Attwood, Tara

2014-10-01

The vitellogenin receptor (Vtgr) plays an important role in fish reproduction. This receptor functions to incorporate vitellogenin (Vtg), a macromolecule synthesized and released from the liver in the bloodstream, into oocytes where it is processed into yolk. Although studies have focused on the functional role of Vtgr in fish, the mechanistic control of this gene is still unexplored. Here we report the identification and analysis of the first piscine 5' regulatory region of the vtgr gene which was cloned from largemouth bass (Micropterus salmoides). Using this putative promoter sequence, we investigated a role for hormones, including insulin and 17β-estradiol (E2), in transcriptional regulation through cell-based reporter assays. No effect of insulin was observed, however, E2 was able to repress transcriptional activity of the vtgr promoter through select estrogen receptor subtypes, Esr1 and Esr2a but not Esr2b. Electrophoretic mobility shift assay demonstrated that Esr1 likely interacts with the vtgr promoter region through half ERE and/or SP1 sites, in part. Finally we also show that ethinylestradiol (EE2), but not bisphenol-A (BPA), represses promoter activity similarly to E2. These results reveal for the first time that the Esr1 isoform may play an inhibitory role in the expression of LMB vtgr mRNA under the influence of E2, and potent estrogens such as EE2. In addition, this new evidence suggests that vtgr may be a target of select endocrine disrupting compounds through environmental exposures. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Transcriptional repression of ER through hMAPK dependent histone deacetylation by class I HDACs.

PubMed

Plotkin, Amy; Volmar, Claude-Henry; Wahlestedt, Claes; Ayad, Nagi; El-Ashry, Dorraya

2014-09-01

Anti-estrogen therapies are not effective in ER- breast cancers, thus identifying mechanisms underlying lack of ER expression in ER- breast cancers is imperative. We have previously demonstrated that hyperactivation of MAPK (hMAPK) downstream of overexpressed EGFR or overexpression/amplification of Her2 represses ER protein and mRNA expression. Abrogation of hMAPK in ER- breast cancer cell lines and primary cultures causes re-expression of ER and restoration of anti-estrogen responses. This study was performed to identify mechanisms of hMAPK-induced transcriptional repression of ER. We found that ER promoter activity is significantly reduced in the presence of hMAPK signaling, yet did not identify specific promoter sequences responsible for this repression. We performed an epigenetic compound screen in an ER- breast cancer cell line that expresses hMAPK yet does not exhibit ER promoter hypermethylation. A number of HDAC inhibitors were identified and confirmed to modulate ER expression and estrogen signaling in multiple ER- cell lines and tumor samples lacking ER promoter methylation. siRNA-mediated knockdown of HDACs 1, 2, and 3 reversed the mRNA repression in multiple breast cancer cell lines and primary cultures and ER promoter-associated histone acetylation increased following MAPK inhibition. These data implicate histone deacetylation downstream of hMAPK in the observed ER mRNA repression associated with hMAPK. Importantly, histone deacetylation appears to be a common mechanism in the transcriptional repression of ER between ER- breast cancers with or without ER promoter hypermethylation.

Kinetically-Defined Component Actions in Gene Repression

PubMed Central

Chow, Carson C.; Finn, Kelsey K.; Storchan, Geoffery B.; Lu, Xinping; Sheng, Xiaoyan; Simons, S. Stoney

2015-01-01

Gene repression by transcription factors, and glucocorticoid receptors (GR) in particular, is a critical, but poorly understood, physiological response. Among the many unresolved questions is the difference between GR regulated induction and repression, and whether transcription cofactor action is the same in both. Because activity classifications based on changes in gene product level are mechanistically uninformative, we present a theory for gene repression in which the mechanisms of factor action are defined kinetically and are consistent for both gene repression and induction. The theory is generally applicable and amenable to predictions if the dose-response curve for gene repression is non-cooperative with a unit Hill coefficient, which is observed for GR-regulated repression of AP1LUC reporter induction by phorbol myristate acetate. The theory predicts the mechanism of GR and cofactors, and where they act with respect to each other, based on how each cofactor alters the plots of various kinetic parameters vs. cofactor. We show that the kinetically-defined mechanism of action of each of four factors (reporter gene, p160 coactivator TIF2, and two pharmaceuticals [NU6027 and phenanthroline]) is the same in GR-regulated repression and induction. What differs is the position of GR action. This insight should simplify clinical efforts to differentially modulate factor actions in gene induction vs. gene repression. PMID:25816223

SAP30L interacts with members of the Sin3A corepressor complex and targets Sin3A to the nucleolus

PubMed Central

Viiri, K. M.; Korkeamäki, H.; Kukkonen, M. K.; Nieminen, L. K.; Lindfors, K.; Peterson, P.; Mäki, M.; Kainulainen, H.; Lohi, O.

2006-01-01

Histone acetylation plays a key role in the regulation of gene expression. The chromatin structure and accessibility of genes to transcription factors is regulated by enzymes that acetylate and deacetylate histones. The Sin3A corepressor complex recruits histone deacetylases and in many cases represses transcription. Here, we report that SAP30L, a close homolog of Sin3-associated protein 30 (SAP30), interacts with several components of the Sin3A corepressor complex. We show that it binds to the PAH3/HID (Paired Amphipathic Helix 3/Histone deacetylase Interacting Domain) region of mouse Sin3A with residues 120–140 in the C-terminal part of the protein. We provide evidence that SAP30L induces transcriptional repression, possibly via recruitment of Sin3A and histone deacetylases. Finally, we characterize a functional nucleolar localization signal in SAP30L and show that SAP30L and SAP30 are able to target Sin3A to the nucleolus. PMID:16820529

Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

PubMed

Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

2000-11-10

In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells. Copyright 2000 Academic Press.

PHYTOCHROME INTERACTING FACTOR3 Associates with the Histone Deacetylase HDA15 in Repression of Chlorophyll Biosynthesis and Photosynthesis in Etiolated Arabidopsis Seedlings[W][OA

PubMed Central

Liu, Xuncheng; Chen, Chia-Yang; Wang, Ko-Ching; Luo, Ming; Tai, Ready; Yuan, Lianyu; Zhao, Minglei; Yang, Songguang; Tian, Gang; Cui, Yuhai; Hsieh, Hsu-Liang; Wu, Keqiang

2013-01-01

PHYTOCHROME INTERACTING FACTOR3 (PIF3) is a key basic helix-loop-helix transcription factor of Arabidopsis thaliana that negatively regulates light responses, repressing chlorophyll biosynthesis, photosynthesis, and photomorphogenesis in the dark. However, the mechanism for the PIF3-mediated transcription regulation remains largely unknown. In this study, we found that the REDUCED POTASSIUM DEPENDENCY3/HISTONE DEACETYLASE1-type histone deacetylase HDA15 directly interacted with PIF3 in vivo and in vitro. Genome-wide transcriptome analysis revealed that HDA15 acts mainly as a transcriptional repressor and negatively regulates chlorophyll biosynthesis and photosynthesis gene expression in etiolated seedlings. HDA15 and PIF3 cotarget to the genes involved in chlorophyll biosynthesis and photosynthesis in the dark and repress gene expression by decreasing the acetylation levels and RNA Polymerase II–associated transcription. The binding of HDA15 to the target genes depends on the presence of PIF3. In addition, PIF3 and HDA15 are dissociated from the target genes upon exposure to red light. Taken together, our results indicate that PIF3 associates with HDA15 to repress chlorophyll biosynthetic and photosynthetic genes in etiolated seedlings. PMID:23548744

Transcriptional Repression of ATF4 Gene by CCAAT/Enhancer-binding Protein β (C/EBPβ) Differentially Regulates Integrated Stress Response*

PubMed Central

Dey, Souvik; Savant, Sudha; Teske, Brian F.; Hatzoglou, Maria; Calkhoven, Cornelis F.; Wek, Ronald C.

2012-01-01

Different environmental stresses induce the phosphorylation of eIF2 (eIF2∼P), repressing global protein synthesis coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in metabolism and nutrient uptake, antioxidation, and regulation of apoptosis. Because ATF4 is a common downstream target that integrates signaling from different eIF2 kinases and their respective stress signals, the eIF2∼P/ATF4 pathway is collectively referred to as the integrated stress response. Although eIF2∼P elicits translational control in response to many different stresses, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. The rationale for this discordant induction of ATF4 expression and eIF2∼P in response to UV irradiation is that transcription of ATF4 is repressed, and therefore ATF4 mRNA is not available for preferential translation. In this study, we show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter, resulting in its transcriptional repression. Expression of C/EBPβ increases in response to UV stress, and the liver-enriched inhibitory protein (LIP) isoform of C/EBPβ, but not the liver-enriched activating protein (LAP) version, represses ATF4 transcription. Loss of the liver-enriched inhibitory protein isoform results in increased ATF4 mRNA levels in response to UV irradiation and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2∼P and translational control combined with transcription factors regulated by alternative signaling pathways can direct programs of gene expression that are specifically tailored to each environmental stress. PMID:22556424

Identification of regulatory targets for the bacterial Nus factor complex.

PubMed

Baniulyte, Gabriele; Singh, Navjot; Benoit, Courtney; Johnson, Richard; Ferguson, Robert; Paramo, Mauricio; Stringer, Anne M; Scott, Ashley; Lapierre, Pascal; Wade, Joseph T

2017-12-11

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

Multiple Genes Repress Motility in Uropathogenic Escherichia coli Constitutively Expressing Type 1 Fimbriae▿ †

PubMed Central

Simms, Amy N.; Mobley, Harry L. T.

2008-01-01

Two surface organelles of uropathogenic Escherichia coli (UPEC), flagella and type 1 fimbriae, are critical for colonization of the urinary tract but mediate opposite actions. Flagella propel bacteria through urine and along mucus layers, while type 1 fimbriae allow bacteria to adhere to specific receptors present on uroepithelial cells. Constitutive expression of type 1 fimbriae leads to repression of motility and chemotaxis in UPEC strain CFT073, suggesting that UPEC may coordinately regulate motility and adherence. To identify genes involved in this regulation of motility by type 1 fimbriae, transposon mutagenesis was performed on a phase-locked type 1 fimbrial ON variant of strain CFT073 (CFT073 fim L-ON), followed by a screen for restoration of motility in soft agar. Functions of the genes identified included attachment, metabolism, transport, DNA mismatch repair, and transcriptional regulation, and a number of genes had hypothetical function. Isogenic deletion mutants of these genes were also constructed in CFT073 fim L-ON. Motility was partially restored in six of these mutants, including complementable mutations in four genes encoding known transcriptional regulators, lrhA, lrp, slyA, and papX; a mismatch repair gene, mutS; and one hypothetical gene, ydiV. Type 1 fimbrial expression in these mutants was unaltered, and the majority of these mutants expressed larger amounts of flagellin than the fim L-ON parental strain. Our results indicate that repression of motility in CFT073 fim L-ON is not solely due to the constitutive expression of type 1 fimbriae on the surfaces of the bacteria and that multiple genes may contribute to this repression. PMID:18359812

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

Transcription Factors in Long-Term Memory and Synaptic Plasticity

PubMed Central

Alberini, Cristina M.

2013-01-01

Transcription is a molecular requisite for long-term synaptic plasticity and long-term memory formation. Thus, in the last several years, one main interest of molecular neuroscience has been the identification of families of transcription factors that are involved in both of these processes. Transcription is a highly regulated process that involves the combined interaction and function of chromatin and many other proteins, some of which are essential for the basal process of transcription, while others control the selective activation or repression of specific genes. These regulated interactions ultimately allow a sophisticated response to multiple environmental conditions, as well as control of spatial and temporal differences in gene expression. Evidence based on correlative changes in expression, genetic mutations, and targeted molecular inhibition of gene expression have shed light on the function of transcription in both synaptic plasticity and memory formation. This review provides a brief overview of experimental work showing that several families of transcription factors, including CREB, C/EBP, Egr, AP-1, and Rel have essential functions in both processes. The results of this work suggest that patterns of transcription regulation represent the molecular signatures of long-term synaptic changes and memory formation. PMID:19126756

NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations.

PubMed

Helmling, Christina; Wacker, Anna; Wolfinger, Michael T; Hofacker, Ivo L; Hengesbach, Martin; Fürtig, Boris; Schwalbe, Harald

2017-02-22

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

PubMed Central

Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

2003-01-01

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

Interlocked feedforward loops control cell-type-specific Rhodopsin expression in the Drosophila eye.

PubMed

Johnston, Robert J; Otake, Yoshiaki; Sood, Pranidhi; Vogt, Nina; Behnia, Rudy; Vasiliauskas, Daniel; McDonald, Elizabeth; Xie, Baotong; Koenig, Sebastian; Wolf, Reinhard; Cook, Tiffany; Gebelein, Brian; Kussell, Edo; Nakagoshi, Hideki; Desplan, Claude

2011-06-10

How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification. Copyright © 2011 Elsevier Inc. All rights reserved.

Transcription factor 7-like 1 is involved in hypothalamo–pituitary axis development in mice and humans

PubMed Central

Gaston-Massuet, Carles; McCabe, Mark J.; Scagliotti, Valeria; Young, Rodrigo M.; Carreno, Gabriela; Gregory, Louise C.; Jayakody, Sujatha A.; Pozzi, Sara; Gualtieri, Angelica; Basu, Basudha; Koniordou, Markela; Wu, Chun-I; Bancalari, Rodrigo E.; Rahikkala, Elisa; Veijola, Riitta; Lopponen, Tuija; Graziola, Federica; Turton, James; Signore, Massimo; Mousavy Gharavy, Seyedeh Neda; Charolidi, Nicoletta; Sokol, Sergei Y.; Merrill, Bradley J.; Dattani, Mehul T.; Martinez-Barbera, Juan Pedro

2016-01-01

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/β-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo–pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke’s pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with β-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH. PMID:26764381

miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis

PubMed Central

Confraria, Ana; Martinho, Cláudia; Elias, Alexandre; Rubio-Somoza, Ignacio; Baena-González, Elena

2013-01-01

The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20–24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response. PMID:23802004

Biochemical and Functional Interactions of Human Papillomavirus Proteins with Polycomb Group Proteins

PubMed Central

McLaughlin-Drubin, Margaret E.; Munger, Karl

2013-01-01

The role of enzymes involved in polycomb repression of gene transcription has been studied extensively in human cancer. Polycomb repressive complexes mediate oncogene-induced senescence, a principal innate cell-intrinsic tumor suppressor pathway that thwarts expansion of cells that have suffered oncogenic hits. Infections with human cancer viruses including human papillomaviruses (HPVs) and Epstein-Barr virus can trigger oncogene-induced senescence, and the viruses have evolved strategies to abrogate this response in order to establish an infection and reprogram their host cells to establish a long-term persistent infection. As a consequence of inhibiting polycomb repression and evading oncogene induced-senescence, HPV infected cells have an altered epigenetic program as evidenced by aberrant homeobox gene expression. Similar alterations are frequently observed in non-virus associated human cancers and may be harnessed for diagnosis and therapy. PMID:23673719

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein

PubMed Central

2014-01-01

Background Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. Results To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug’s target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Conclusions Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism. PMID:24393533

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

PubMed

Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

2014-01-07

Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

The Initiation of Epigenetic Silencing of Active Transposable Elements Is Triggered by RDR6 and 21-22 Nucleotide Small Interfering RNAs1[W][OA

PubMed Central

Nuthikattu, Saivageethi; McCue, Andrea D.; Panda, Kaushik; Fultz, Dalen; DeFraia, Christopher; Thomas, Erica N.; Slotkin, R. Keith

2013-01-01

Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genome-wide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing. PMID:23542151

The initiation of epigenetic silencing of active transposable elements is triggered by RDR6 and 21-22 nucleotide small interfering RNAs.

PubMed

Nuthikattu, Saivageethi; McCue, Andrea D; Panda, Kaushik; Fultz, Dalen; DeFraia, Christopher; Thomas, Erica N; Slotkin, R Keith

2013-05-01

Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genome-wide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing.

The crystal structure of the AhRR-ARNT heterodimer reveals the structural basis of the repression of AhR-mediated transcription.

PubMed

Sakurai, Shunya; Shimizu, Toshiyuki; Ohto, Umeharu

2017-10-27

2,3,7,8-Tetrachlorodibenzo- p -dioxin and related compounds are extraordinarily potent environmental toxic pollutants. Most of the 2,3,7,8-tetrachlorodibenzo- p -dioxin toxicities are mediated by aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor belonging to the basic helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) family. Upon ligand binding, AhR forms a heterodimer with AhR nuclear translocator (ARNT) and induces the expression of genes involved in various biological responses. One of the genes induced by AhR encodes AhR repressor (AhRR), which also forms a heterodimer with ARNT and represses the activation of AhR-dependent transcription. The control of AhR activation is critical for managing AhR-mediated diseases, but the mechanisms by which AhRR represses AhR activation remain poorly understood, because of the lack of structural information. Here, we determined the structure of the AhRR-ARNT heterodimer by X-ray crystallography, which revealed an asymmetric intertwined domain organization presenting structural features that are both conserved and distinct among bHLH-PAS family members. The structures of AhRR-ARNT and AhR-ARNT were similar in the bHLH-PAS-A region, whereas the PAS-B of ARNT in the AhRR-ARNT complex exhibited a different domain arrangement in this family reported so far. The structure clearly disclosed that AhRR competitively represses AhR binding to ARNT and target DNA and further suggested the existence of an AhRR-ARNT-specific repression mechanism. This study provides a structural basis for understanding the mechanism by which AhRR represses AhR-mediated gene transcription. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Opposing PKA and Hog1 signals control the post-transcriptional response to glucose availability in Cryptococcus neoformans.

PubMed

Banerjee, Dithi; Bloom, Amanda L M; Panepinto, John C

2016-10-01

The pathogenic fungus Cryptococcus neoformans must adapt to glucose-limited conditions in the lung and glucose replete conditions upon dissemination to the brain. We report that glucose controls ribosome biogenesis and translation by modulating mRNA decay through a balance of PKA and Hog1 signalling. Glucose signalling through PKA stabilized ribosomal protein (RP) mRNAs whereas glucose starvation destabilized RP transcripts through Hog1. Glucose starvation-induced oxidative stress response genes, and treatment of glucose-fed cells with reactive oxygen species (ROS) generating compounds repressed RP transcripts, both of which were dependent on Hog1. Stabilization of RP transcripts led to retention of polysomes in a hog1Δ mutant, whereas stabilization of RP transcripts by cyclic AMP did not affect translation repression, suggesting that Hog1 alone signals translation repression. In sum, this work describes a novel antagonism between PKA and Hog1 controlling ribosome biogenesis via mRNA stability in response to glucose availability in this important human pathogen. © 2016 John Wiley & Sons Ltd.

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.

Repression of ESR1 transcription by MYOD potentiates letrozole-resistance in ERα-positive breast cancer cells.

PubMed

Zhang, Qiang; Liu, Xiao-Yan; Li, Shuang; Zhao, Zhao; Li, Juan; Cui, Ming-Ke; Wang, En-Hua

2017-10-21

Transcriptional silencing of estrogen receptor α (ERα) expression is an important etiology contributing to the letrozole-resistance in ERα-positive breast cancer (BCa) cells, but the transcription factors responsible for this transcriptional repression remain largely unidentified. Here we report that the expression of the basic helix-loop-helix myogenic regulatory factor MYOD was abnormally up-regulated in letrozole-resistant BCa tissues and in experimentally-induced letrozole-resistant BCa cells. Overexpression of the exogenous MYOD impaired ERα expression and potentiated letrozole-resistance in letrozole-sensitive MCF7 cells, whereas MYOD knockdown could effectively restore ERα expression and thereby promote letrozole-sensitivity in letrozole-resistant MCF-7/LR cells. Mechanistically, MYOD was shown to be a potent corepressor of ESR1 transcription, and this transcriptional repression was significantly enhanced in the presence of letrozole treatment. Thus, targeted inhibition of MYOD may restore ERα level and lead to resensitization to letrozole-based hormone therapy, providing a novel therapeutic strategy for relapsed ERα-positive BCa patients. Our data also underscore an unexpected chemotherapeutic facet of MYOD, which may operate as a novel regulator of BCa biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences.

PubMed

Steel, L F; Telly, D L; Leonard, J; Rice, B A; Monks, B; Sawicki, J A

1996-10-01

Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF. Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs). To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region. Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR. We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs. Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect. The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation. Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

Maintaining Sufficient Nanos Is a Critical Function for Polar Granule Component in the Specification of Primordial Germ Cells

PubMed Central

Deshpande, Girish; Spady, Emma; Goodhouse, Joe; Schedl, Paul

2012-01-01

Primordial germ cells (PGC) are the precursors of germline stem cells. In Drosophila, PGC specification is thought to require transcriptional quiescence and three genes, polar granule component (pgc), nanos (nos), and germ cell less (gcl) function to downregulate Pol II transcription. While it is not understood how nos or gcl represses transcription, pgc does so by inhibiting the transcription elongation factor b (P-TEFb), which is responsible for phosphorylating Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of the largest Pol II subunit. In the studies reported here, we demonstrate that nos are a critical regulatory target of pgc. We show that a substantial fraction of the PGCs in pgc embryos have greatly reduced levels of Nos protein and exhibit phenotypes characteristic of nos PGCs. Lastly, restoring germ cell–specific expression of Nos is sufficient to ameliorate the pgc phenotype. PMID:23173091

Maintaining sufficient nanos is a critical function for polar granule component in the specification of primordial germ cells.

PubMed

Deshpande, Girish; Spady, Emma; Goodhouse, Joe; Schedl, Paul

2012-11-01

Primordial germ cells (PGC) are the precursors of germline stem cells. In Drosophila, PGC specification is thought to require transcriptional quiescence and three genes, polar granule component (pgc), nanos (nos), and germ cell less (gcl) function to downregulate Pol II transcription. While it is not understood how nos or gcl represses transcription, pgc does so by inhibiting the transcription elongation factor b (P-TEFb), which is responsible for phosphorylating Ser2 residues in the heptad repeat of the C-terminal domain (CTD) of the largest Pol II subunit. In the studies reported here, we demonstrate that nos are a critical regulatory target of pgc. We show that a substantial fraction of the PGCs in pgc embryos have greatly reduced levels of Nos protein and exhibit phenotypes characteristic of nos PGCs. Lastly, restoring germ cell-specific expression of Nos is sufficient to ameliorate the pgc phenotype.

Transcriptional and post-transcriptional regulation of NK cell development and function

PubMed Central

Leong, Jeffrey W.; Wagner, Julia A.; Ireland, Aaron R.; Fehniger, Todd A.

2016-01-01

Natural killer (NK) cells are specialized innate lymphoid cells that survey against viral infections and malignancy. Numerous advances have improved our understanding of the molecular mechanisms that control NK cell development and function over the past decade. These include both studies on the regulatory effects of transcription factors and translational repression via microRNAs. In this review, we summarize our current knowledge of DNA-binding transcription factors that regulate gene expression and thereby orchestrate NK cell development and activation, with an emphasis on recent discoveries. Additionally, we highlight our understanding of how RNA-bindings microRNAs fine tune the NK cell molecular program. We also underscore the large number of open questions in field that are now being addressed using new technological approaches and genetically engineered model organisms. Ultimately, a deeper understanding of the basic molecular biology of NK cells will facilitate new strategies to manipulate NK cells for the treatment of human disease. PMID:26948928

The Pseudomonas aeruginosa AmrZ C-terminal domain mediates tetramerization and is required for its activator and repressor functions

PubMed Central

Xu, Binjie; Ju, Yue; Soukup, Randal J.; Ramsey, Deborah M.; Fishel, Richard; Wysocki, Vicki H.; Wozniak, Daniel J.

2015-01-01

Summary Pseudomonas aeruginosa is an important bacterial opportunistic pathogen, presenting a significant threat towards individuals with underlying diseases such as cystic fibrosis. The transcription factor AmrZ regulates expression of multiple P. aeruginosa virulence factors. AmrZ belongs to the ribbon-helix-helix protein superfamily, in which many members function as dimers, yet others form higher-order oligomers. In this study, four independent approaches were undertaken and demonstrated that the primary AmrZ form in solution is tetrameric. Deletion of the AmrZ C-terminal domain leads to loss of tetramerization and reduced DNA binding to both activated and repressed target promoters. Additionally, the C-terminal domain is essential for efficient AmrZ-mediated activation and repression of its targets. PMID:26549743

Cuf2 Is a Novel Meiosis-Specific Regulatory Factor of Meiosis Maturation

PubMed Central

Ioannoni, Raphael; Beaudoin, Jude; Lopez-Maury, Luis; Codlin, Sandra; Bahler, Jurg; Labbe, Simon

2012-01-01

Background Meiosis is the specialized form of the cell cycle by which diploid cells produce the haploid gametes required for sexual reproduction. Initiation and progression through meiosis requires that the expression of the meiotic genes is precisely controlled so as to provide the correct gene products at the correct times. During meiosis, four temporal gene clusters are either induced or repressed by a cascade of transcription factors. Principal Findings In this report a novel copper-fist-type regulator, Cuf2, is shown to be expressed exclusively during meiosis. The expression profile of the cuf2+ mRNA revealed that it was induced during middle-phase meiosis. Both cuf2+ mRNA and protein levels are unregulated by copper addition or starvation. The transcription of cuf2+ required the presence of a functional mei4+ gene encoding a key transcription factor that activates the expression of numerous middle meiotic genes. Microscopic analyses of cells expressing a functional Cuf2-GFP protein revealed that Cuf2 co-localized with both homologous chromosomes and sister chromatids during the meiotic divisions. Cells lacking Cuf2 showed an elevated and sustained expression of several of the middle meiotic genes that persisted even during late meiosis. Moreover, cells carrying disrupted cuf2Δ/cuf2Δ alleles displayed an abnormal morphology of the forespore membranes and a dramatic reduction of spore viability. Significance Collectively, the results revealed that Cuf2 functions in the timely repression of the middle-phase genes during meiotic differentiation. PMID:22558440

The bHLH transcription factor GmPIB1 facilitates resistance to Phytophthora sojae in Glycine max

PubMed Central

Cheng, Qun; Dong, Lidong; Gao, Tianjiao; Liu, Tengfei; Li, Ninghui; Wang, Le; Chang, Xin; Wu, Junjiang; Xu, Pengfei

2018-01-01

Abstract Phytophthora sojae Kaufmann and Gerdemann causes Phytophthora root rot, a destructive soybean disease worldwide. A basic helix–loop–helix (bHLH) transcription factor is thought to be involved in the response to P. sojae infection in soybean, as revealed by RNA sequencing (RNA-seq). However, the molecular mechanism underlying this response is currently unclear. Here, we explored the function and underlying mechanisms of a bHLH transcription factor in soybean, designated GmPIB1 (P. sojae-inducible bHLH transcription factor), during host responses to P. sojae. GmPIB1 was significantly induced by P. sojae in the resistant soybean cultivar ‘L77-1863’. Analysis of transgenic soybean hairy roots with elevated or reduced expression of GmPIB1 demonstrated that GmPIB1 enhances resistance to P. sojae and reduces reactive oxygen species (ROS) accumulation. Quantitative reverse transcription PCR and chromatin immunoprecipitation–quantitative PCR assays revealed that GmPIB1 binds directly to the promoter of GmSPOD1 and represses its expression; this gene encodes a key enzyme in ROS production. Moreover, transgenic soybean hairy roots with GmSPOD1 silencing through RNA interference exhibited improved resistance to P. sojae and reduced ROS generation. These findings suggest that GmPIB1 enhances resistance to P. sojae by repressing the expression of GmSPOD1. PMID:29579245

Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription.

PubMed

Stapleton, P C; Dobson, A D W

2003-04-25

Cellobiose dehydrogenase (CDH) production in Trametes versicolor is induced in the presence of cellulose, but decreases when additional carbon sources such as glucose and maltose are added to the fungal cultures. Using T. versicolor-specific cdh primers in a reverse transcription-polymerase chain reaction-based approach, it appears that this repression in CDH production is being mediated at the level of gene transcription. When a 1.6-kb upstream region of the T. versicolor cdh gene was cloned and sequenced, a number of putative CreA-like binding sites were observed. We propose that these sites may be involved in mediating this repressive effect, based on their similarity to the consensus [5'-SYGGRGG-3'] site for binding of the CreA and Cre1 repressor proteins.

Genome expression analysis in yeast reveals novel transcriptional regulation by inositol and choline and new regulatory functions for Opi1p, Ino2p, and Ino4p.

PubMed

Santiago, Teresa C; Mamoun, Choukri Ben

2003-10-03

In Saccharomyces cerevisiae, genes encoding phospholipid-synthesizing enzymes are regulated by inositol and choline (IC). The current model suggests that when these precursors become limiting, the transcriptional complex Ino2p-Ino4p activates the expression of these genes, whereas repression requires Opi1p and occurs when IC are available. In this study, microarray-based expression analysis was performed to assess the global transcriptional response to IC in a wild-type strain and in the opi1delta, ino2delta, and ino4delta null mutant strains. Fifty genes were either activated or repressed by IC in the wild-type strain, including three already known IC-repressed genes. We demonstrated that the IC response was not limited to genes involved in membrane biogenesis, but encompassed various metabolic pathways such as biotin synthesis, one-carbon compound metabolism, nitrogen-containing compound transport and degradation, cell wall organization and biogenesis, and acetyl-CoA metabolism. The expression of a large number of IC-regulated genes did not change in the opi1delta, ino2delta, and ino4delta strains, thus implicating new regulatory elements in the IC response. Our studies revealed that Opi1p, Ino2p, and Ino4p have dual regulatory activities, acting in both positive and negative transcriptional regulation of a large number of genes, most of which are not regulated by IC and only a subset of which is involved in membrane biogenesis. These data provide the first global response profile of yeast to IC and reveal novel regulatory mechanisms by these precursors.

Opposite Smad and chicken ovalbumin upstream promoter transcription factor inputs in the regulation of the collagen VII gene promoter by transforming growth factor-beta.

PubMed

Calonge, María Julia; Seoane, Joan; Massagué, Joan

2004-05-28

A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-beta (TGF-beta). The gene, COL7A1, is activated by TGF-beta via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-beta may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-beta-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-beta-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-beta-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-beta addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-beta signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs.

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bossi, Flavia; Fan, Jue; Xiao, Jun

Here, the molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. As a result, to identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation.more » We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.« less

Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction

DOE PAGES

Bossi, Flavia; Fan, Jue; Xiao, Jun; ...

2017-06-26

Here, the molecular function of a gene is most commonly inferred by sequence similarity. Therefore, genes that lack sufficient sequence similarity to characterized genes (such as certain classes of transcriptional regulators) are difficult to classify using most function prediction algorithms and have remained uncharacterized. As a result, to identify novel transcriptional regulators systematically, we used a feature-based pipeline to screen protein families of unknown function. This method predicted 43 transcriptional regulator families in Arabidopsis thaliana, 7 families in Drosophila melanogaster, and 9 families in Homo sapiens. Literature curation validated 12 of the predicted families to be involved in transcriptional regulation.more » We tested 33 out of the 195 Arabidopsis putative transcriptional regulators for their ability to activate transcription of a reporter gene in planta and found twelve coactivators, five of which had no prior literature support. To investigate mechanisms of action in which the predicted regulators might work, we looked for interactors of an Arabidopsis candidate that did not show transactivation activity in planta and found that it might work with other members of its own family and a subunit of the Polycomb Repressive Complex 2 to regulate transcription. Our results demonstrate the feasibility of assigning molecular function to proteins of unknown function without depending on sequence similarity. In particular, we identified novel transcriptional regulators using biological features enriched in transcription factors. The predictions reported here should accelerate the characterization of novel regulators.« less

CIP, a cardiac Isl1-interacting protein, represses cardiomyocyte hypertrophy

PubMed Central

Huang, Zhan-Peng; Seok, Hee Young; Zhou, Bin; Chen, Jinghai; Chen, Jian-Fu; Tao, Yazhong; Pu, William T.; Wang, Da-Zhi

2012-01-01

Rationale Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well established, the molecular events that inhibit or repress cardiac hypertrophy are less known. Objective To identify and investigate novel regulators that modulate cardiac hypertrophy. Methods and Results Here, we report the identification, characterization and functional examination of CIP, a novel cardiac Isl1-interacting protein. CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast-two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a co-factor of CIP. CIP directly interacted with Isl1 and we mapped the domains of these two proteins which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the MEF2C enhancer. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Conclusions Our studies therefore identify CIP a novel regulator of cardiac hypertrophy. PMID:22343712

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae.

PubMed

Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

2016-07-08

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Hsp70 homolog Ssb and the 14-3-3 protein Bmh1 jointly regulate transcription of glucose repressed genes in Saccharomyces cerevisiae

PubMed Central

Hübscher, Volker; Mudholkar, Kaivalya; Chiabudini, Marco; Fitzke, Edith; Wölfle, Tina; Pfeifer, Dietmar; Drepper, Friedel; Warscheid, Bettina; Rospert, Sabine

2016-01-01

Chaperones of the Hsp70 family interact with a multitude of newly synthesized polypeptides and prevent their aggregation. Saccharomyces cerevisiae cells lacking the Hsp70 homolog Ssb suffer from pleiotropic defects, among others a defect in glucose-repression. The highly conserved heterotrimeric kinase SNF1/AMPK (AMP-activated protein kinase) is required for the release from glucose-repression in yeast and is a key regulator of energy balance also in mammalian cells. When glucose is available the phosphatase Glc7 keeps SNF1 in its inactive, dephosphorylated state. Dephosphorylation depends on Reg1, which mediates targeting of Glc7 to its substrate SNF1. Here we show that the defect in glucose-repression in the absence of Ssb is due to the ability of the chaperone to bridge between the SNF1 and Glc7 complexes. Ssb performs this post-translational function in concert with the 14-3-3 protein Bmh, to which Ssb binds via its very C-terminus. Raising the intracellular concentration of Ssb or Bmh enabled Glc7 to dephosphorylate SNF1 even in the absence of Reg1. By that Ssb and Bmh efficiently suppressed transcriptional deregulation of Δreg1 cells. The findings reveal that Ssb and Bmh comprise a new chaperone module, which is involved in the fine tuning of a phosphorylation-dependent switch between respiration and fermentation. PMID:27001512

Transcription of fgf8 is regulated by activating and repressive cis-elements at the midbrain-hindbrain boundary in zebrafish embryos.

PubMed

Inoue, Fumitaka; Parvin, Mst Shahnaj; Yamasu, Kyo

2008-04-15

Fgf8 is expressed in the isthmic region of the developing brain, serving an organizing function in vertebrate embryos. We previously identified S4.2 downstream to the zebrafish fgf8 gene as a regulatory region that drives transcription in the anterior hindbrain. Here, we investigated the mechanism of fgf8 regulation by the S4.2 region during development. Reporter analyses in embryos revealed that S4.2 closely recapitulates fgf8 expression in the anteriormost hindbrain during somitogenesis. This region contains a sequence highly conserved in fgf8 of diverse vertebrates. Further analyses of S4.2 revealed a 342-bp core region composed of three subregions (#2, #3, and #4). Regions #3 and #4 drove expression broadly in the brain from the midbrain to r5 of the hindbrain, whereas a 28-bp sequence in #2 repressed ectopic expression in the midbrain and in r2 to r5. The enhancer function of S4.2 was absent in pax2a mutant embryos, while it was activated ectopically by pax2a misexpression in the hindbrain. We identified two sites in the core region that are bound by Pax2a in vitro and in vivo, the disruption of which abrogated the S4.2 activity. Thus, fgf8 expression in the anteriormost hindbrain involves activation and repression, with Pax2a as a pivotal regulator.

DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans

PubMed Central

Amrit, Francis Raj Gandhi; Steenkiste, Elizabeth Marie; Ratnappan, Ramesh; Chen, Shaw-Wen; McClendon, T. Brooke; Kostka, Dennis; Yanowitz, Judith; Olsen, Carissa Perez; Ghazi, Arjumand

2016-01-01

Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation. PMID:26862916

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

Dynamic Repositioning of Dorsal to Two Different κB Motifs Controls Its Autoregulation during Immune Response in Drosophila

PubMed Central

Mrinal, Nirotpal; Nagaraju, Javaregowda

2010-01-01

Autoregulation is one of the mechanisms of imparting feedback control on gene expression. Positive autoregulatory feedback results in induction of a gene, and negative feedback leads to its suppression. Here, we report an interesting mechanism of autoregulation operating on Drosophila Rel gene dorsal that can activate as well as repress its expression. Using biochemical and genetic approaches, we show that upon immune challenge Dorsal regulates its activation as well as repression by dynamically binding to two different κB motifs, κBI (intronic κB) and κBP (promoter κB), present in the dorsal gene. Although the κBI motif functions as an enhancer, the κBP motif acts as a transcriptional repressor. Interestingly, Dorsal binding to these two motifs is dynamic; immediately upon immune challenge, Dorsal binds to the κBI leading to auto-activation, whereas at the terminal phase of the immune response, it is removed from the κBI and repositioned at the κBP, resulting in its repression. Furthermore, we show that repression of Dorsal as well as its binding to the κBP depends on the transcription factor AP1. Depletion of AP1 by RNA interference resulted in constitutive expression of Dorsal. In conclusion, this study suggests that during acute phase response dorsal is regulated by following two subcircuits: (i) Dl-κBI for activation and (ii) Dl-AP1-κBP for repression. These two subcircuits are temporally delineated and bring about overall regulation of dorsal during immune response. These results suggest the presence of a previously unknown mechanism of Dorsal autoregulation in immune-challenged Drosophila. PMID:20504768

pH-Dependent DNA Distortion and Repression of Gene Expression by Pectobacterium atrosepticum PecS.

PubMed

Deochand, Dinesh K; Meariman, Jacob K; Grove, Anne

2016-07-15

Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the relative positioning of -10 and -35 promoter elements. The plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes, must alter gene expression patterns to ensure growth in planta. In the related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression. Here, we report that P. atrosepticum PecS controls gene activity by altering promoter DNA topology in response to pH. While PecS binds the pecS promoter with high affinity regardless of pH, it induces significant DNA distortion only at neutral pH, the pH at which the pecS promoter is repressed in vivo. At pH ∼8, DNA distortions are attenuated, and PecS no longer represses the pecS promoter. A specific histidine (H142) located in a crevice between the dimerization- and DNA-binding regions is required for pH-dependent changes in DNA distortion and repression of gene activity, and mutation of this histidine renders the mutant protein incapable of repressing the pecS promoter. We propose that protonated PecS induces a DNA conformation at neutral pH in which -10 and -35 promoter elements are suboptimally positioned for RNA polymerase binding; on deprotonation of PecS, binding is no longer associated with significant changes in DNA conformation, allowing gene expression. We suggest that this mode of gene regulation leads to differential expression of the PecS regulon in response to alkalinization of the plant apoplast.

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila

PubMed Central

Kwok, Rosanna S.; Li, Ying H.; Lei, Anna J.; Edery, Isaac; Chiu, Joanna C.

2015-01-01

Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription. PMID:26132408

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

PubMed

Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

PubMed Central

Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription.

PubMed

Muth, V; Nadaud, S; Grummt, I; Voit, R

2001-03-15

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

The Budding Yeast Nucleus

PubMed Central

Taddei, Angela; Schober, Heiko; Gasser, Susan M.

2010-01-01

The budding yeast nucleus, like those of other eukaryotic species, is highly organized with respect to both chromosomal sequences and enzymatic activities. At the nuclear periphery interactions of nuclear pores with chromatin, mRNA, and transport factors promote efficient gene expression, whereas centromeres, telomeres, and silent chromatin are clustered and anchored away from pores. Internal nuclear organization appears to be function-dependent, reflecting localized sites for tRNA transcription, rDNA transcription, ribosome assembly, and DNA repair. Recent advances have identified new proteins involved in the positioning of chromatin and have allowed testing of the functional role of higher-order chromatin organization. The unequal distribution of silent information regulatory factors and histone modifying enzymes, which arises in part from the juxtaposition of telomeric repeats, has been shown to influence chromatin-mediated transcriptional repression. Other localization events suppress unwanted recombination. These findings highlight the contribution budding yeast genetics and cytology have made to dissecting the functional role of nuclear structure. PMID:20554704

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression

PubMed Central

Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A.; Ramaswamy, Suresh; Plant, Tony M.; Ojeda, Sergio R.

2015-01-01

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty. PMID:26671628

Glucocorticoid Repression of Inflammatory Gene Expression Shows Differential Responsiveness by Transactivation- and Transrepression-Dependent Mechanisms

PubMed Central

King, Elizabeth M.; Chivers, Joanna E.; Rider, Christopher F.; Minnich, Anne; Giembycz, Mark A.; Newton, Robert

2013-01-01

Binding of glucocorticoid to the glucocorticoid receptor (GR/NR3C1) may repress inflammatory gene transcription via direct, protein synthesis-independent processes (transrepression), or by activating transcription (transactivation) of multiple anti-inflammatory/repressive factors. Using human pulmonary A549 cells, we showed that 34 out of 39 IL-1β-inducible mRNAs were repressed to varying degrees by the synthetic glucocorticoid, dexamethasone. Whilst these repressive effects were GR-dependent, they did not correlate with either the magnitude of IL-1β-inducibility or the NF-κB-dependence of the inflammatory genes. This suggests that induction by IL-1β and repression by dexamethasone are independent events. Roles for transactivation were investigated using the protein synthesis inhibitor, cycloheximide. However, cycloheximide reduced the IL-1β-dependent expression of 13 mRNAs, which, along with the 5 not showing repression by dexamethasone, were not analysed further. Of the remaining 21 inflammatory mRNAs, cycloheximide significantly attenuated the dexamethasone-dependent repression of 11 mRNAs that also showed a marked time-dependence to their repression. Such effects are consistent with repression occurring via the de novo synthesis of a new product, or products, which subsequently cause repression (i.e., repression via a transactivation mechanism). Conversely, 10 mRNAs showed completely cycloheximide-independent, and time-independent, repression by dexamethasone. This is consistent with direct GR transrepression. Importantly, the inflammatory mRNAs showing attenuated repression by dexamethasone in the presence of cycloheximide also showed a significantly greater extent of repression and a higher potency to dexamethasone compared to those mRNAs showing cycloheximide-independent repression. This suggests that the repression of inflammatory mRNAs by GR transactivation-dependent mechanisms accounts for the greatest levels of repression and the most potent repression by dexamethasone. In conclusion, our data indicate roles for both transrepression and transactivation in the glucocorticoid-dependent repression of inflammatory gene expression. However, transactivation appears to account for the more potent and efficacious mechanism of repression by glucocorticoids on these IL-1β-induced genes. PMID:23349769

The Arabidopsis bZIP11 transcription factor links low-energy signalling to auxin-mediated control of primary root growth

PubMed Central

Weiste, Christoph; Pedrotti, Lorenzo; Muralidhara, Prathibha; Ljung, Karin; Dröge-Laser, Wolfgang

2017-01-01

Plants have to tightly control their energy homeostasis to ensure survival and fitness under constantly changing environmental conditions. Thus, it is stringently required that energy-consuming stress-adaptation and growth-related processes are dynamically tuned according to the prevailing energy availability. The evolutionary conserved SUCROSE NON-FERMENTING1 RELATED KINASES1 (SnRK1) and the downstream group C/S1 basic leucine zipper (bZIP) transcription factors (TFs) are well-characterised central players in plants’ low-energy management. Nevertheless, mechanistic insights into plant growth control under energy deprived conditions remains largely elusive. In this work, we disclose the novel function of the low-energy activated group S1 bZIP11-related TFs as regulators of auxin-mediated primary root growth. Whereas transgenic gain-of-function approaches of these bZIPs interfere with the activity of the root apical meristem and result in root growth repression, root growth of loss-of-function plants show a pronounced insensitivity to low-energy conditions. Based on ensuing molecular and biochemical analyses, we propose a mechanistic model, in which bZIP11-related TFs gain control over the root meristem by directly activating IAA3/SHY2 transcription. IAA3/SHY2 is a pivotal negative regulator of root growth, which has been demonstrated to efficiently repress transcription of major auxin transport facilitators of the PIN-FORMED (PIN) gene family, thereby restricting polar auxin transport to the root tip and in consequence auxin-driven primary root growth. Taken together, our results disclose the central low-energy activated SnRK1-C/S1-bZIP signalling module as gateway to integrate information on the plant’s energy status into root meristem control, thereby balancing plant growth and cellular energy resources. PMID:28158182

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

DOE PAGES

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna; ...

2018-05-25

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

The Arabidopsis bZIP11 transcription factor links low-energy signalling to auxin-mediated control of primary root growth.

PubMed

Weiste, Christoph; Pedrotti, Lorenzo; Selvanayagam, Jebasingh; Muralidhara, Prathibha; Fröschel, Christian; Novák, Ondřej; Ljung, Karin; Hanson, Johannes; Dröge-Laser, Wolfgang

2017-02-01

Plants have to tightly control their energy homeostasis to ensure survival and fitness under constantly changing environmental conditions. Thus, it is stringently required that energy-consuming stress-adaptation and growth-related processes are dynamically tuned according to the prevailing energy availability. The evolutionary conserved SUCROSE NON-FERMENTING1 RELATED KINASES1 (SnRK1) and the downstream group C/S1 basic leucine zipper (bZIP) transcription factors (TFs) are well-characterised central players in plants' low-energy management. Nevertheless, mechanistic insights into plant growth control under energy deprived conditions remains largely elusive. In this work, we disclose the novel function of the low-energy activated group S1 bZIP11-related TFs as regulators of auxin-mediated primary root growth. Whereas transgenic gain-of-function approaches of these bZIPs interfere with the activity of the root apical meristem and result in root growth repression, root growth of loss-of-function plants show a pronounced insensitivity to low-energy conditions. Based on ensuing molecular and biochemical analyses, we propose a mechanistic model, in which bZIP11-related TFs gain control over the root meristem by directly activating IAA3/SHY2 transcription. IAA3/SHY2 is a pivotal negative regulator of root growth, which has been demonstrated to efficiently repress transcription of major auxin transport facilitators of the PIN-FORMED (PIN) gene family, thereby restricting polar auxin transport to the root tip and in consequence auxin-driven primary root growth. Taken together, our results disclose the central low-energy activated SnRK1-C/S1-bZIP signalling module as gateway to integrate information on the plant's energy status into root meristem control, thereby balancing plant growth and cellular energy resources.

Repression of myoblast proliferation and fibroblast growth factor receptor 1 promoter activity by KLF10 protein.

PubMed

Parakati, Rajini; DiMario, Joseph X

2013-05-10

FGFR1 gene expression regulates myoblast proliferation and differentiation, and its expression is controlled by Krüppel-like transcription factors. KLF10 interacts with the FGFR1 promoter, repressing its activity and cell proliferation. KLF10 represses FGFR1 promoter activity and thereby myoblast proliferation. A model of transcriptional control of chicken FGFR1 gene regulation during myogenesis is presented. Skeletal muscle development is controlled by regulation of myoblast proliferation and differentiation into muscle fibers. Growth factors such as fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation and differentiation in numerous tissues, including skeletal muscle. Transcriptional regulation of FGFR1 gene expression is developmentally regulated by the Sp1 transcription factor, a member of the Krüppel-like factor (KLF) family of transcriptional regulators. Here, we show that another KLF transcription factor, KLF10, also regulates myoblast proliferation and FGFR1 promoter activity. Expression of KLF10 reduced myoblast proliferation by 86%. KLF10 expression also significantly reduced FGFR1 promoter activity in myoblasts and Sp1-mediated FGFR1 promoter activity in Drosophila SL2 cells. Southwestern blot, electromobility shift, and chromatin immunoprecipitation assays demonstrated that KLF10 bound to the proximal Sp factor binding site of the FGFR1 promoter and reduced Sp1 complex formation with the FGFR1 promoter at that site. These results indicate that KLF10 is an effective repressor of myoblast proliferation and represses FGFR1 promoter activity in these cells via an Sp1 binding site.

Transcription Factor Foxo1 Is a Negative Regulator of NK Cell Maturation and Function

PubMed Central

Deng, Youcai; Kerdiles, Yann; Chu, Jianhong; Yuan, Shunzong; Wang, Youwei; Chen, Xilin; Mao, Hsiaoyin; Zhang, Lingling; Zhang, Jianying; Hughes, Tiffany; Deng, Yafei; Zhang, Qi; Wang, Fangjie; Zou, Xianghong; Liu, Chang-Gong; Freud, Aharon G.; Li, Xiaohui; Caligiuri, Michael A; Vivier, Eric; Yu, Jianhua

2015-01-01

SUMMARY Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes through upregulating CD62L expression, and impaired late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21+/− mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions. PMID:25769609

CRISPR-mediated HDAC2 disruption identifies two distinct classes of target genes in human cells.

PubMed

Somanath, Priyanka; Herndon Klein, Rachel; Knoepfler, Paul S

2017-01-01

The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.

Direct interactions of OCA-B and TFII-I regulate immunoglobulin heavy-chain gene transcription by facilitating enhancer-promoter communication.

PubMed

Ren, Xiaodi; Siegel, Rachael; Kim, Unkyu; Roeder, Robert G

2011-05-06

B cell-specific coactivator OCA-B, together with Oct-1/2, binds to octamer sites in promoters and enhancers to activate transcription of immunoglobulin (Ig) genes, although the mechanisms underlying their roles in enhancer-promoter communication are unknown. Here, we demonstrate a direct interaction of OCA-B with transcription factor TFII-I, which binds to DICE elements in Igh promoters, that affects transcription at two levels. First, OCA-B relieves HDAC3-mediated Igh promoter repression by competing with HDAC3 for binding to promoter-bound TFII-I. Second, and most importantly, Igh 3' enhancer-bound OCA-B and promoter-bound TFII-I mediate promoter-enhancer interactions, in both cis and trans, that are important for Igh transcription. These and other results reveal an important function for OCA-B in Igh 3' enhancer function in vivo and strongly favor an enhancer mechanism involving looping and facilitated factor recruitment rather than a tracking mechanism. Copyright © 2011 Elsevier Inc. All rights reserved.

Direct interactions of OCA-B and TFII-I regulate immunoglobulin heavy-chain gene transcription by facilitating enhancer-promoter communication

PubMed Central

Ren, Xiaodi; Siegel, Rachael; Kim, Unkyu; Roeder, Robert G.

2011-01-01

Summary B cell-specific coactivator OCA-B, together with Oct-1/2, binds to octamer sites in promoters and enhancers to activate transcription of immunoglobulin (Ig) genes, although the mechanisms underlying their roles in enhancer-promoter communication are unknown. Here, we demonstrate a direct interaction of OCA-B with transcription factor TFII-I, which binds to DICE elements in IgH promoters, that affects transcription at two levels. First, OCA-B relieves HDAC3-mediated IgH promoter repression by competing with HDAC3 for binding to promoter-bound TFII-I. Second, and most importantly, Igh 3′enhancer-bound OCA-B and promoter-bound TFII-I mediate promoter-enhancer interactions, in both cis and trans, that are important for Igh transcription. These and other results reveal an important function for OCA-B in Igh 3′enhancer function in vivo and strongly favor an enhancer mechanism involving looping and facilitated factor recruitment rather than a tracking mechanism. PMID:21549311

Functional interrogation of non-coding DNA through CRISPR genome editing

PubMed Central

Canver, Matthew C.; Bauer, Daniel E.; Orkin, Stuart H.

2017-01-01

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. PMID:28288828

Regulation of Root Greening by Light and Auxin/Cytokinin Signaling in Arabidopsis[W

PubMed Central

Kobayashi, Koichi; Baba, Shinsuke; Obayashi, Takeshi; Sato, Mayuko; Toyooka, Kiminori; Keränen, Mika; Aro, Eva-Mari; Fukaki, Hidehiro; Ohta, Hiroyuki; Sugimoto, Keiko; Masuda, Tatsuru

2012-01-01

Tight coordination between plastid differentiation and plant development is best evidenced by the synchronized development of photosynthetic tissues and the biogenesis of chloroplasts. Here, we show that Arabidopsis thaliana roots demonstrate accelerated chlorophyll accumulation and chloroplast development when they are detached from shoots. However, this phenomenon is repressed by auxin treatment. Mutant analyses suggest that auxin transported from the shoot represses root greening via the function of INDOLE-3-ACETIC ACID14, AUXIN RESPONSE FACTOR7 (ARF7), and ARF19. Cytokinin signaling, on the contrary, is required for chlorophyll biosynthesis in roots. The regulation by auxin/cytokinin is dependent on the transcription factor LONG HYPOCOTYL5 (HY5), which is required for the expression of key chlorophyll biosynthesis genes in roots. The expression of yet another root greening transcription factor, GOLDEN2-LIKE2 (GLK2), was found to be regulated in opposing directions by auxin and cytokinin. Furthermore, both the hormone signaling and the GLK transcription factors modified the accumulation of HY5 in roots. Overexpression of GLKs in the hy5 mutant provided evidence that GLKs require HY5 to maximize their activities in root greening. We conclude that the combination of HY5 and GLKs, functioning downstream of light and auxin/cytokinin signaling pathways, is responsible for coordinated expression of the key genes in chloroplast biogenesis. PMID:22415275

Overexpression of OsGATA12 regulates chlorophyll content, delays plant senescence and improves rice yield under high density planting.

PubMed

Lu, Guangwen; Casaretto, José A; Ying, Shan; Mahmood, Kashif; Liu, Fang; Bi, Yong-Mei; Rothstein, Steven J

2017-05-01

Agronomic traits controlling the formation, architecture and physiology of source and sink organs are main determinants of rice productivity. Semi-dwarf rice varieties with low tiller formation but high seed production per panicle and dark green and thick leaves with prolonged source activity are among the desirable traits to further increase the yield potential of rice. Here, we report the functional characterization of a zinc finger transcription factor, OsGATA12, whose overexpression causes increased leaf greenness, reduction of leaf and tiller number, and affects yield parameters. Reduced tillering allowed testing the transgenic plants under high density which resulted in significantly increased yield per area and higher harvest index compared to wild-type. We show that delayed senescence of transgenic plants and the corresponding longer stay-green phenotype is mainly due to increased chlorophyll and chloroplast number. Further, our work postulates that the increased greenness observed in the transgenic plants is due to more chlorophyll synthesis but most significantly to decreased chlorophyll degradation, which is supported by the reduced expression of genes involved in the chlorophyll degradation pathway. In particular we show evidence for the down-regulation of the STAY GREEN RICE gene and in vivo repression of its promoter by OsGATA12, which suggests a transcriptional repression function for a GATA transcription factor for prolonging the onset of senescence in cereals.

Jasmonate Regulates the INDUCER OF CBF EXPRESSION–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 Cascade and Freezing Tolerance in Arabidopsis[W

PubMed Central

Hu, Yanru; Jiang, Liqun; Wang, Fang; Yu, Diqiu

2013-01-01

The INDUCER OF CBF EXPRESSION (ICE)–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several JASMONATE ZIM-DOMAIN (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance. PMID:23933884

Jasmonate regulates the inducer of cbf expression-C-repeat binding factor/DRE binding factor1 cascade and freezing tolerance in Arabidopsis.

PubMed

Hu, Yanru; Jiang, Liqun; Wang, Fang; Yu, Diqiu

2013-08-01

The inducer of cbf expression (ICE)-C-repeat binding factor/DRE binding factor1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several jasmonate ZIM-domain (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance.

Corticosteroids inhibit sphingosine 1-phosphate-induced interleukin-6 secretion from human airway smooth muscle via mitogen-activated protein kinase phosphatase 1-mediated repression of mitogen and stress-activated protein kinase 1.

PubMed

Che, Wenchi; Parmentier, Johannes; Seidel, Petra; Manetsch, Melanie; Ramsay, Emma E; Alkhouri, Hatem; Ge, Qi; Armour, Carol L; Ammit, Alaina J

2014-02-01

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that plays an important proinflammatory role in asthmatic airways. Corticosteroids are first-line antiinflammatories in asthma; however, their repressive effects on S1P-induced cytokine secretion have not been investigated. To address this, our in vitro study reveals the molecular mechanisms by which corticosteroids inhibit S1P-induced IL-6 expression in the pivotal immunomodulatory cell type, airway smooth muscle (ASM). We first uncover the cellular signaling pathways responsible: S1P activates a cyclic adenosine monophosphate/cAMP response-element-binding protein (CREB)/CRE-dependent pathway to induce IL-6 transcription, concomitant with stimulation of the mitogen-activated protein kinase (MAPK) superfamily and downstream mitogen and stress-activated protein kinase 1 (MSK1) and histone H3 phosphorylation. In this way, S1P stimulates parallel signaling pathways to induce IL-6 secretion via CRE-driven transcription of the IL-6 gene promoter in a relaxed chromatin environment achieved through histone H3 phosphorylation. Second, we investigated how corticosteroids mediate their repressive effects. The corticosteroid dexamethasone inhibits S1P-induced IL-6 protein secretion and mRNA expression, but CREB/CRE transrepression, inhibition of IL-6 mRNA stability, or subcellular relocation of MSK1 were not responsible for the repressive effects of dexamethasone. Rather, we show that dexamethasone rapidly induces up-regulation of the MAPK deactivator MAPK phosphatase 1 (MKP-1) and that MKP-1 blocks the MAPK-driven activation of MSK1 and phosphorylation of histone H3. This was confirmed by treatment with triptolide, an inhibitor of MKP-1 up-regulation, where repressive effects of corticosteroids were reversed. Our study reveals the molecular mechanism underlying the antiinflammatory capacity of corticosteroids to repress proinflammatory functions induced by the potent bioactive sphingolipid S1P in the lung.

The Epstein-Barr Virus Episome Maneuvers between Nuclear Chromatin Compartments during Reactivation

PubMed Central

Moquin, Stephanie A.; Thomas, Sean; Whalen, Sean; Warburton, Alix; Fernandez, Samantha G.; McBride, Alison A.; Pollard, Katherine S.

2017-01-01

ABSTRACT The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation. IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription. PMID:29142137

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

Roles of CAMTA transcription factors and salicylic acid in configuring the low-temperature transcriptome and freezing tolerance of Arabidopsis.

PubMed

Kim, YongSig; Park, Sunchung; Gilmour, Sarah J; Thomashow, Michael F

2013-08-01

Previous studies in Arabidopsis thaliana established roles for CALMODULIN BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) in the rapid cold induction of CRT/DRE BINDING FACTOR (CBF) genes CBF1 and CBF2, and the repression of salicylic acid (SA) biosynthesis at warm temperature. Here we show that CAMTA1 and CAMTA2 work in concert with CAMTA3 at low temperature (4°C) to induce peak transcript levels of CBF1, CBF2 and CBF3 at 2 h, contribute to up-regulation of approximately 15% of the genes induced at 24 h, most of which fall outside the CBF pathway, and increase plant freezing tolerance. In addition, CAMTA1, CAMTA2 and CAMTA3 function together to inhibit SA biosynthesis at warm temperature (22°C). However, SA levels increase in Arabidopsis plants that are exposed to low temperature for more than 1 week. We show that this chilling-induced SA biosynthesis proceeds through the isochorismate synthase (ICS) pathway, with cold induction of ICS1 (which encodes ICS), and two genes encoding transcription factors that positively regulate ICS1 - CBP60g and SARD1 -, paralleling SA accumulation. The three CAMTA proteins effectively repress the accumulation of ICS1, CBP60g and SARD1 transcripts at warm temperature but not at low temperature. This impairment of CAMTA function may involve post-transcriptional regulation, as CAMTA transcript levels did not decrease at low temperature. Salicylic acid biosynthesis at low temperature did not contribute to freezing tolerance, but had a major role in configuring the transcriptome, including the induction of 'defense response' genes, suggesting the possible existence of a pre-emptive defense strategy programmed by prolonged chilling temperatures. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Ski co-repressor complexes maintain the basal repressed state of the TGF-beta target gene, SMAD7, via HDAC3 and PRMT5.

PubMed

Tabata, Takanori; Kokura, Kenji; Ten Dijke, Peter; Ishii, Shunsuke

2009-01-01

The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-beta-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-beta target gene, in TGF-beta-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-beta-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.

Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during Drosophila hematopoiesis

PubMed Central

Gobert, Vanessa; Augé, Benoit; Burlet-Schiltz, Odile; Haenlin, Marc

2017-01-01

A tight regulation of transcription factor activity is critical for proper development. For instance, modifications of RUNX transcription factors dosage are associated with several diseases, including hematopoietic malignancies. In Drosophila, Myeloid Leukemia Factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (Lz). However, the mechanism of action of this conserved family of proteins involved in leukemia remains largely unknown. Here we further characterized MLF’s mode of action in Drosophila blood cells using proteomic, transcriptomic and genetic approaches. Our results show that MLF and the Hsp40 co-chaperone family member DnaJ-1 interact through conserved domains and we demonstrate that both proteins bind and stabilize Lz in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex that directly regulates Lz activity. Importantly, dnaj-1 loss causes an increase in Lz+ blood cell number and size similarly as in mlf mutant larvae. Moreover we find that dnaj-1 genetically interacts with mlf to control Lz level and Lz+ blood cell development in vivo. In addition, we show that mlf and dnaj-1 loss alters Lz+ cell differentiation and that the increase in Lz+ blood cell number and size observed in these mutants is caused by an overactivation of the Notch signaling pathway. Finally, using different conditions to manipulate Lz activity, we show that high levels of Lz are required to repress Notch transcription and signaling. All together, our data indicate that the MLF/DnaJ-1-dependent increase in Lz level allows the repression of Notch expression and signaling to prevent aberrant blood cell development. Thus our findings establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development in vivo. PMID:28742844

HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse

PubMed Central

Lim, Shu Ly; Geoghegan, Joel; Hempfling, Anna-Lena; Bergmann, Martin; Goodnow, Christopher C.; Ormandy, Christopher J.; Wong, Lee; Mann, Jeff; Scott, Hamish S.; Jamsai, Duangporn; Adelson, David L.

2015-01-01

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program. PMID:26496356

The Two Cryptochrome/Photolyase Family Proteins Fulfill Distinct Roles in DNA Photorepair and Regulation of Conidiation in the Gray Mold Fungus Botrytis cinerea

PubMed Central

Cohrs, Kim C.

2017-01-01

ABSTRACT The plant-pathogenic leotiomycete Botrytis cinerea is known for the strict regulation of its asexual differentiation programs by environmental light conditions. Sclerotia are formed in constant darkness; black/near-UV (NUV) light induces conidiation; and blue light represses both differentiation programs. Sensing of black/NUV light is attributed to proteins of the cryptochrome/photolyase family (CPF). To elucidate the molecular basis of the photoinduction of conidiation, we functionally characterized the two CPF proteins encoded in the genome of B. cinerea as putative positive-acting components. B. cinerea CRY1 (BcCRY1), a cyclobutane pyrimidine dimer (CPD) photolyase, acts as the major enzyme of light-driven DNA repair (photoreactivation) and has no obvious role in signaling. In contrast, BcCRY2, belonging to the cry-DASH proteins, is dispensable for photorepair but performs regulatory functions by repressing conidiation in white and especially black/NUV light. The transcription of bccry1 and bccry2 is induced by light in a White Collar complex (WCC)-dependent manner, but neither light nor the WCC is essential for the repression of conidiation through BcCRY2 when bccry2 is constitutively expressed. Further, BcCRY2 affects the transcript levels of both WCC-induced and WCC-repressed genes, suggesting a signaling function downstream of the WCC. Since both CPF proteins are dispensable for photoinduction by black/NUV light, the origin of this effect remains elusive and may be connected to a yet unknown UV-light-responsive system. IMPORTANCE Botrytis cinerea is an economically important plant pathogen that causes gray mold diseases in a wide variety of plant species, including high-value crops and ornamental flowers. The spread of disease in the field relies on the formation of conidia, a process that is regulated by different light qualities. While this feature has been known for a long time, we are just starting to understand the underlying molecular mechanisms. Conidiation in B. cinerea is induced by black/near-UV light, whose sensing is attributed to the action of cryptochrome/photolyase family (CPF) proteins. Here we report on the distinct functions of two CPF proteins in the photoresponse of B. cinerea. While BcCRY1 acts as the major photolyase in photoprotection, BcCRY2 acts as a cryptochrome with a signaling function in regulating photomorphogenesis (repression of conidiation). PMID:28667107

Characterization of BreR Interaction with the Bile Response Promoters breAB and breR in Vibrio cholerae

PubMed Central

Cerda-Maira, Francisca A.; Kovacikova, Gabriela; Jude, Brooke A.; Skorupski, Karen

2013-01-01

The Vibrio cholerae BreR protein is a transcriptional repressor of the breAB efflux system operon, which encodes proteins involved in bile resistance. In a previous study (F. A. Cerda-Maira, C. S. Ringelberg, and R. K. Taylor, J. Bacteriol. 190:7441–7452, 2008), we used gel mobility shift assays to determine that BreR binds at two independent binding sites at the breAB promoter and a single site at its own promoter. Here it is shown, by DNase I footprinting and site-directed mutagenesis, that BreR is able to bind at a distal and a proximal site in the breAB promoter. However, only one of these sites, the proximal 29-bp site, is necessary for BreR-mediated transcriptional repression of breAB expression. In addition, it was determined that BreR represses its own expression by recognizing a 28-bp site at the breR promoter. These sites comprise regions of dyad symmetry within which residues critical for BreR function could be identified. The BreR consensus sequence AANGTANAC-N6-GTNTACNTT overlaps the −35 region at both promoters, implying that the repression of gene expression is achieved by interfering with RNA polymerase binding at these promoters. PMID:23144245

A p53-inducible microRNA-34a downregulates Ras signaling by targeting IMPDH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hwa-Ryeon; Roe, Jae-Seok; Lee, Ji-Eun

2012-02-24

Highlights: Black-Right-Pointing-Pointer p53 downregulates IMPDH. Black-Right-Pointing-Pointer p53-dependent miR-34a transactivation inhibits IMPDH transcription. Black-Right-Pointing-Pointer miR-34a-mediated inhibition of IMPDH downregulates GTP-dependent Ras signal. -- Abstract: p53 is a well-known transcription factor that controls cell cycle arrest and cell death in response to a wide range of stresses. Moreover, p53 regulates glucose metabolism and its mutation results in the metabolic switch to the Warburg effect found in cancer cells. Nucleotide biosynthesis is also critical for cell proliferation and the cell division cycle. Nonetheless, little is known about whether p53 regulates nucleotide biosynthesis. Here we demonstrated that p53-inducible microRNA-34a (miR-34a) repressed inosine 5 Primemore » -monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme of de novo GTP biosynthesis. Treatment with anti-miR-34a inhibitor relieved the expression of IMPDH upon DNA damage. Ultimately, miR-34a-mediated inhibition of IMPDH resulted in repressed activation of the GTP-dependent Ras signaling pathway. In summary, we suggest that p53 has a novel function in regulating purine biosynthesis, aided by miR-34a-dependent IMPDH repression.« less

Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression

PubMed Central

Luo, Michelle L.; Mullis, Adam S.; Leenay, Ryan T.; Beisel, Chase L.

2015-01-01

CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing and transcriptional regulation. Because these RNA-directed immune systems are found in most prokaryotes, an opportunity exists to harness the endogenous systems as convenient tools in these organisms. Here, we report that the Type I-E CRISPR-Cas system in Escherichia coli can be co-opted for programmable transcriptional repression. We found that deletion of the signature cas3 gene converted this immune system into a programmable gene regulator capable of reversible gene silencing of heterologous and endogenous genes. Targeting promoter regions yielded the strongest repression, whereas targeting coding regions showed consistent strand bias. Furthermore, multi-targeting CRISPR arrays could generate complex phenotypes. This strategy offers a simple approach to convert many endogenous Type I systems into transcriptional regulators, thereby expanding the available toolkit for CRISPR-mediated genetic control while creating new opportunities for genome-wide screens and pathway engineering. PMID:25326321

NFIB-mediated repression of the epigenetic factor Ezh2 regulates cortical development.

PubMed

Piper, Michael; Barry, Guy; Harvey, Tracey J; McLeay, Robert; Smith, Aaron G; Harris, Lachlan; Mason, Sharon; Stringer, Brett W; Day, Bryan W; Wray, Naomi R; Gronostajski, Richard M; Bailey, Timothy L; Boyd, Andrew W; Richards, Linda J

2014-02-19

Epigenetic mechanisms are essential in regulating neural progenitor cell self-renewal, with the chromatin-modifying protein Enhancer of zeste homolog 2 (EZH2) emerging as a central player in promoting progenitor cell self-renewal during cortical development. Despite this, how Ezh2 is itself regulated remains unclear. Here, we demonstrate that the transcription factor nuclear factor IB (NFIB) plays a key role in this process. Nfib(-/-) mice exhibit an increased number of proliferative ventricular zone cells that express progenitor cell markers and upregulation of EZH2 expression within the neocortex and hippocampus. NFIB binds to the Ezh2 promoter and overexpression of NFIB represses Ezh2 transcription. Finally, key downstream targets of EZH2-mediated epigenetic repression are misregulated in Nfib(-/-) mice. Collectively, these results suggest that the downregulation of Ezh2 transcription by NFIB is an important component of the process of neural progenitor cell differentiation during cortical development.

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

PubMed Central

Takahashi, Melissa K.; Watters, Kyle E.; Gasper, Paul M.; Abbott, Timothy R.; Carlson, Paul D.; Chen, Alan A.

2016-01-01

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure–function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure–function design principles for a diverse array of natural and synthetic RNA regulators. PMID:27103533

Runx1t1 (Runt-Related Transcription Factor 1; Translocated to, 1) Epigenetically Regulates the Proliferation and Nitric Oxide Production of Microglia

PubMed Central

Baby, Nimmi; Li, Yali; Ling, Eng-Ang; Lu, Jia; Dheen, S. Thameem

2014-01-01

Background Microglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions. Methods and Findings siRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2. Conclusion/Interpretation In conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs. PMID:24586690

CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase

PubMed Central

Park, Hongmarn; Yakhnin, Helen; Connolly, Michael; Romeo, Tony

2015-01-01

ABSTRACT Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5′ untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5′ fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation. IMPORTANCE CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity. PMID:26438818

The Transcriptional Regulator, CosR, Controls Compatible Solute Biosynthesis and Transport, Motility and Biofilm Formation in Vibrio cholerae

PubMed Central

Shikuma, Nicholas J.; Davis, Kimberly R.; Fong, Jiunn N. C.; Yildiz, Fitnat H.

2012-01-01

SUMMARY Vibrio cholerae inhabits aquatic environments and colonizes the human digestive tract to cause the disease cholera. In these environments, V. cholerae copes with fluctuations in salinity and osmolarity by producing and transporting small, organic, highly soluble molecules called compatible solutes, which counteract extracellular osmotic pressure. Currently, it is unclear how V. cholerae regulates the expression of genes important for the biosynthesis or transport of compatible solutes in response to changing salinity or osmolarity conditions. Through a genome-wide transcriptional analysis of the salinity response of V. cholerae, we identified a transcriptional regulator we name CosR for compatible solute regulator. The expression of cosR is regulated by ionic strength and not osmolarity. A transcriptome analysis of a ΔcosR mutant revealed that CosR represses genes involved in ectoine biosynthesis and compatible solute transport in a salinity-dependent manner. When grown in salinities similar to estuarine environments, CosR activates biofilm formation and represses motility independently of its function as an ectoine regulator. This is the first study to characterize a compatible solute regulator in V. cholerae and couples the regulation of osmotic tolerance with biofilm formation and motility. PMID:22690884

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

Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A

PubMed Central

Zhou, Lei; Le Roux, Gwenaëlle; Ducrot, Cécile; Chédin, Stéphane; Labarre, Jean; Riva, Michel; Carles, Christophe

2013-01-01

Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd2+) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd2+ rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd2+, but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I–Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag+ and Hg2+, which likewise perturb the Pol I–Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I–Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals. PMID:23640330

Long noncoding RNA EWSAT1-mediated gene repression facilitates Ewing sarcoma oncogenesis

PubMed Central

Marques Howarth, Michelle; Simpson, David; Ngok, Siu P.; Nieves, Bethsaida; Chen, Ron; Siprashvili, Zurab; Vaka, Dedeepya; Breese, Marcus R.; Crompton, Brian D.; Alexe, Gabriela; Hawkins, Doug S.; Jacobson, Damon; Brunner, Alayne L.; West, Robert; Mora, Jaume; Stegmaier, Kimberly; Khavari, Paul; Sweet-Cordero, E. Alejandro

2014-01-01

Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma–associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes. PMID:25401475

Cyclin D1 in the Liver: Role of Noncanonical Signaling in Liver Steatosis and Hormone Regulation

PubMed Central

Núñez, Kelley G.; Gonzalez-Rosario, Janet; Thevenot, Paul T.; Cohen, Ari J.

2017-01-01

Background: Cyclin D1 is an important protein for cell cycle progression; however, functions independent of the cell cycle have been described in the liver. Cyclin D1 is also involved in DNA repair, is overexpressed in many cancers, and functions as a proto-oncogene. The lesser-known roles of Cyclin D1, specifically in hepatocytes, impact liver steatosis and hormone regulation in the liver. Methods: A comprehensive search of PubMed was conducted using the keywords Cyclin D1, steatosis, lipogenesis, and liver transplantation. In this article, we review the results from this literature search, with a focus on the role of Cyclin D1 in hepatic lipogenesis and gluconeogenesis, as well as the impact and function of this protein in hepatic steatosis. Results: Cyclin D1 represses carbohydrate response element binding protein (ChREBP) and results in a decrease in transcription of fatty acid synthase (FAS) and acetyl-coenzyme A carboxylase (ACC). Cyclin D1 also inhibits peroxisome proliferator-activated receptor gamma (PPARγ) which is involved in hepatic lipogenesis. Cyclin D1 inhibits both hepatocyte nuclear factor 4 alpha (HNF4α) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) and represses transcription of lipogenic genes FAS and liver-type pyruvate kinase (Pklr), along with the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Conclusion: Cyclin D1 represses multiple proteins involved in both lipogenesis and gluconeogenesis in the liver. Targeting Cyclin D1 to decrease hepatic steatosis in patients with nonalcoholic fatty liver disease or alcoholic fatty liver disease may help improve patient health and the quality of the donor liver pool. PMID:28331449

A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells.

PubMed

Zhang, Zhong-Lin; Shin, Margaret; Zou, Xiaolu; Huang, Jianzhi; Ho, Tun-hua David; Shen, Qingxi J

2009-05-01

Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.

GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis

PubMed Central

Breuer, Christian; Kawamura, Ayako; Clark, Natalie M.; Morohashi, Kengo; Busch, Wolfgang; Benfey, Philip N.; Sozzani, Rosangela

2018-01-01

ABSTRACT How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis. Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors. PMID:29439132

Microchidia protein 2, MORC2, downregulates the cytoskeleton adapter protein, ArgBP2, via histone methylation in gastric cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong, Yuxin; Li, Yan; Gu, Hui

ArgBP2 is an adapter protein that plays an important role in actin-dependent processes such as cell adhesion and migration. However, its function and regulation mechanisms in gastric cancer have not yet been investigated. Here, we showed the low expression of ArgBP2 mRNA level in gastric tumor samples and its repressive function in the proliferation, migration, and invasion of gastric cancer cells. Then, we cloned and identified ArgBP2 promoter and verified that MORC2 bound to the promoter. Moreover, we demonstrated that MORC2 enhanced the recruitment of EZH2, which promoted the tri-methylation of H3K27, leading to the transcriptional repression of ArgBP2. Ourmore » results might thus contribute to understanding the molecular mechanisms of ArgBP2 regulation and suggesting ArgBP2 as a potential therapeutic target for gastric cancer. - Highlights: • ArgBP2 inhibits proliferation, migration, and invasion of gastric cancer cells. • Identification of ArgBP2 promoter and its transcription factor MORC2. • EZH2 is required in MORC2 down-regulating ArgBP2 via histone methylation.« less

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

PubMed Central

Nazer, Ezequiel; Dale, Ryan K.; Chinen, Madoka; Radmanesh, Behram

2018-01-01

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression. PMID:29529026

Curcumin differentially regulates endoplasmic reticulum stress through transcriptional corepressor SMILE (small heterodimer partner-interacting leucine zipper protein)-mediated inhibition of CREBH (cAMP responsive element-binding protein H).

PubMed

Misra, Jagannath; Chanda, Dipanjan; Kim, Don-kyu; Li, Tiangang; Koo, Seung-Hoi; Back, Sung-Hoon; Chiang, John Y L; Choi, Hueng-Sik

2011-12-09

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), is a natural polyphenolic compound. Herein the effect of curcumin on endoplasmic reticulum (ER) stress responsive gene expression was investigated. We report that curcumin induces transcriptional corepressor small heterodimer partner-interacting leucine zipper protein (SMILE) gene expression through liver kinase B1 (LKB1)/adenosine monophosphate-activated kinase (AMPK) signaling pathway and represses ER stress-responsive gene transcription in an ER-bound transcription factor specific manner. cAMP responsive element-binding protein H (CREBH) and activating transcription factor 6 (ATF6) are both ER-bound bZIP family transcription factors that are activated upon ER stress. Of interest, we observed that both curcumin treatment and SMILE overexpression only represses CREBH-mediated transactivation of the target gene but not ATF6-mediated transactivation. Knockdown of endogenous SMILE significantly releases the inhibitory effect of curcumin on CREBH transactivation. Intrinsic repressive activity of SMILE is observed in the Gal4 fusion system, and the intrinsic repressive domain is mapped to the C terminus of SMILE spanning amino acid residues 203-269, corresponding to the basic region leucine zipper (bZIP) domain. In vivo interaction assay revealed that through its bZIP domain, SMILE interacts with CREBH and inhibits its transcriptional activity. Interestingly, we observed that SMILE does not interact with ATF6. Furthermore, competition between SMILE and the coactivator peroxisome proliferator-activated receptor α (PGC-1α) on CREBH transactivation has been demonstrated in vitro and in vivo. Finally, chromatin immunoprecipitation assays revealed that curcumin decreases the binding of PGC-1α and CREBH on target gene promoter in a SMILE-dependent manner. Overall, for the first time we suggest a novel phenomenon that the curcumin/LKB1/AMPK/SMILE/PGC1α pathway differentially regulates ER stress-mediated gene transcription.

Thyroid hormone and COUP-TF1 regulate kallikrein-binding protein (KBP) gene expression.

PubMed

Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen; Brent, Gregory A

2011-03-01

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T(3) and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5' flanking region (-53 to -29) and nTRE2, located in the first intron (104-132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T(3). COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T(3) repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T(3). Nuclear corepressor knockdown resulted in loss of T(3) repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T(3) induction of positive thyroid hormone response elements, reverses T(3) repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T(3) and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T(3).

Thyroid Hormone and COUP-TF1 Regulate Kallikrein-Binding Protein (KBP) Gene Expression

PubMed Central

Liu, Yan-Yun; Nakatani, Teruyo; Kogai, Takahiko; Mody, Kaizeen

2011-01-01

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T3 and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5′ flanking region (−53 to −29) and nTRE2, located in the first intron (104–132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T3. COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T3 repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T3. Nuclear corepressor knockdown resulted in loss of T3 repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T3 induction of positive thyroid hormone response elements, reverses T3 repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T3 and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T3. PMID:21266512

Trithorax dependent changes in chromatin landscape at enhancer and promoter regions drive female puberty.

PubMed

Toro, Carlos A; Wright, Hollis; Aylwin, Carlos F; Ojeda, Sergio R; Lomniczi, Alejandro

2018-01-04

Polycomb group (PcG) proteins control the timing of puberty by repressing the Kiss1 gene in hypothalamic arcuate nucleus (ARC) neurons. Here we identify two members of the Trithorax group (TrxG) of modifiers, mixed-lineage leukemia 1 (MLL1), and 3 (MLL3), as central components of an activating epigenetic machinery that dynamically counteracts PcG repression. Preceding puberty, MLL1 changes the chromatin configuration at the promoters of Kiss1 and Tac3, two genes required for puberty to occur, from repressive to permissive. Concomitantly, MLL3 institutes a chromatin structure that changes the functional status of a Kiss1 enhancer from poised to active. RNAi-mediated, ARC-specific Mll1 knockdown reduced Kiss1 and Tac3 expression, whereas CRISPR-Cas9-directed epigenome silencing of the Kiss1 enhancer selectively reduced Kiss1 activity. Both interventions delay puberty and disrupt reproductive cyclicity. Our results demonstrate that an epigenetic switch from transcriptional repression to activation is crucial to the regulatory mechanism controlling the timing of mammalian puberty.

Characterization of a JAZ7 activation-tagged Arabidopsis mutant with increased susceptibility to the fungal pathogen Fusarium oxysporum

PubMed Central

Thatcher, Louise F.; Cevik, Volkan; Grant, Murray; Zhai, Bing; Jones, Jonathan D.G.; Manners, John M.; Kazan, Kemal

2016-01-01

In Arabidopsis, jasmonate (JA)-signaling plays a key role in mediating Fusarium oxysporum disease outcome. However, the roles of JASMONATE ZIM-domain (JAZ) proteins that repress JA-signaling have not been characterized in host resistance or susceptibility to this pathogen. Here, we found most JAZ genes are induced following F. oxysporum challenge, and screening T-DNA insertion lines in Arabidopsis JAZ family members identified a highly disease-susceptible JAZ7 mutant (jaz7-1D). This mutant exhibited constitutive JAZ7 expression and conferred increased JA-sensitivity, suggesting activation of JA-signaling. Unlike jaz7 loss-of-function alleles, jaz7-1D also had enhanced JA-responsive gene expression, altered development and increased susceptibility to the bacterial pathogen Pst DC3000 that also disrupts host JA-responses. We also demonstrate that JAZ7 interacts with transcription factors functioning as activators (MYC3, MYC4) or repressors (JAM1) of JA-signaling and contains a functional EAR repressor motif mediating transcriptional repression via the co-repressor TOPLESS (TPL). We propose through direct TPL recruitment, in wild-type plants JAZ7 functions as a repressor within the JA-response network and that in jaz7-1D plants, misregulated ectopic JAZ7 expression hyper-activates JA-signaling in part by disturbing finely-tuned COI1-JAZ-TPL-TF complexes. PMID:26896849

Glucocorticoid Receptor-Mediated Repression of Pro-Inflammatory Genes in Rheumatoid Arthritis

DTIC Science & Technology

2015-10-01

1 AWARD NUMBER: W81XWH-14-1-0314 TITLE: Glucocorticoid Receptor-Mediated Repression of Pro-Inflammatory Genes in Rheumatoid Arthritis ...19 Sep 2015 4. TITLE AND SUBTITLE Glucocorticoid Receptor-Mediated Repression of Pro- Inflammatory Genes in Rheumatoid Arthritis 5a. CONTRACT NUMBER...SUBJECT TERMS Rheumatoid arthritis , inflammation and autoimmunity, macrophages, glucocorticoid receptor, transcriptional regulation, coactivators and

Different Levels of Catabolite Repression Optimize Growth in Stable and Variable Environments

PubMed Central

New, Aaron M.; Cerulus, Bram; Govers, Sander K.; Perez-Samper, Gemma; Zhu, Bo; Boogmans, Sarah; Xavier, Joao B.; Verstrepen, Kevin J.

2014-01-01

Organisms respond to environmental changes by adapting the expression of key genes. However, such transcriptional reprogramming requires time and energy, and may also leave the organism ill-adapted when the original environment returns. Here, we study the dynamics of transcriptional reprogramming and fitness in the model eukaryote Saccharomyces cerevisiae in response to changing carbon environments. Population and single-cell analyses reveal that some wild yeast strains rapidly and uniformly adapt gene expression and growth to changing carbon sources, whereas other strains respond more slowly, resulting in long periods of slow growth (the so-called “lag phase”) and large differences between individual cells within the population. We exploit this natural heterogeneity to evolve a set of mutants that demonstrate how the frequency and duration of changes in carbon source can favor different carbon catabolite repression strategies. At one end of this spectrum are “specialist” strategies that display high rates of growth in stable environments, with more stringent catabolite repression and slower transcriptional reprogramming. The other mutants display less stringent catabolite repression, resulting in leaky expression of genes that are not required for growth in glucose. This “generalist” strategy reduces fitness in glucose, but allows faster transcriptional reprogramming and shorter lag phases when the cells need to shift to alternative carbon sources. Whole-genome sequencing of these mutants reveals that mutations in key regulatory genes such as HXK2 and STD1 adjust the regulation and transcriptional noise of metabolic genes, with some mutations leading to alternative gene regulatory strategies that allow “stochastic sensing” of the environment. Together, our study unmasks how variable and stable environments favor distinct strategies of transcriptional reprogramming and growth. PMID:24453942

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

PubMed

Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

2016-02-01

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. Copyright © 2016 by the Genetics Society of America.

Profligate biotin synthesis in α-proteobacteria - a developing or degenerating regulatory system?

PubMed

Feng, Youjun; Zhang, Huimin; Cronan, John E

2013-04-01

Biotin (vitamin H) is a key enzyme cofactor required in all three domains of life. Although this cofactor was discovered over 70 years ago and has long been recognized as an essential nutrient for animals, our knowledge of the strategies bacteria use to sense biotin demand is very limited. The paradigm mechanism is that of Escherichia coli in which BirA protein, the prototypical bi-functional biotin protein ligase, both covalently attaches biotin to the acceptor proteins of central metabolism and represses transcription of the biotin biosynthetic pathway in response to biotin demand. However, in other bacteria the biotin protein ligase lacks a DNA-binding domain which raises the question of how these bacteria regulate the synthesis of biotin, an energetically expensive molecule. A bioinformatic study by Rodionov and Gelfand identified a protein termed BioR in α-proteobacteria and predicted that BioR would have the biotin operon regulatory role that in most other bacteria is fulfilled by the BirA DNA-binding domain. We have now tested this prediction in the plant pathogen Agrobacterium tumefaciens. As predicted the A. tumefaciens biotin protein ligase is a fully functional ligase that has no role in regulation of biotin synthesis whereas BioR represses transcription of the biotin synthesis genes. Moreover, as determined by electrophoretic mobility shift assays, BioR binds the predicted operator site, which is located downstream of the mapped transcription start site. qPCR measurements indicated that deletion of BioR resulted in a c. 15-fold increase of bio operon transcription in the presence of high biotin levels. Effective repression of a plasmid-borne bioB-lacZ reporter was seen only upon the overproduction of BioR. In contrast to E. coli and Bacillus subtilis where biotin synthesis is tightly controlled, A. tumefaciens synthesizes much more biotin than needed for modification of the biotin-requiring enzymes. Protein-bound biotin constitutes only about 0.5% of the total biotin, most of which is found in the culture medium. To the best of our knowledge, A. tumefaciens represents the first example of profligate biotin synthesis by a wild type bacterium. © 2013 Blackwell Publishing Ltd.

Repression of the Low Affinity Iron Transporter Gene FET4

PubMed Central

Caetano, Soraia M.; Menezes, Regina; Amaral, Catarina; Rodrigues-Pousada, Claudina; Pimentel, Catarina

2015-01-01

Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake. PMID:26063801

First report of an unusual novel double mutation affecting the transcription repression domain of MeCP2 and causing a severe phenotype of Rett syndrome: Molecular analyses and computational investigation.

PubMed

Ghorbel, Rania; Ghorbel, Raouia; Rouissi, Aida; Fendri-Kriaa, Nourhene; Ben Salah, Ghada; Belguith, Neila; Ammar-Keskes, Leila; Gouider-Khouja, Neziha; Fakhfakh, Faiza

2018-02-26

Rett syndrome is an X-linked neurodevelopmental disorder that develops a profound intellectual and motor disability and affects 1 from 10 000 to 15 000 live female births. This disease is characterized by a period of apparently normal development until 6-18 months of age when motor and communication abilities regress which is caused by mutations occurred in the X-linked MECP2 gene, encoding the methyl-CpG binding protein 2. This research study reports a molecular analysis via an exhaustive gene sequencing which reveals an unusual novel double mutation (c.695 G > T; c.880C > T) located in a highly conserved region in MECP2 gene affecting the transcription repression domain (TRD) of MeCP2 protein and leading for the first time to a severe phenotype of Rett syndrome. Moreover, a computational investigation of MECP2 mutations demonstrates that the novel mutation c.695 G > T is highly deleterious which affects the MeCP2 protein showing also an adverse impact on MECP2 gene expression and resulting in an affected folding and decreased stability of MECP2 structures. Thus, the altered TRD domain engenders a disrupted process of MECP2 functions. Therefore, this is the first study which highlights a novel double mutation among the transcription repression domain (TRD) of MeCP2 protein in Rett patient with a severe clinical phenotype in North Africa region. Copyright © 2018 Elsevier Inc. All rights reserved.

CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia.

PubMed

Hampton, Hannah G; McNeil, Matthew B; Paterson, Thomas J; Ney, Blair; Williamson, Neil R; Easingwood, Richard A; Bostina, Mihnea; Salmond, George P C; Fineran, Peter C

2016-06-01

SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.

CRISPR-Cas gene-editing reveals RsmA and RsmC act through FlhDC to repress the SdhE flavinylation factor and control motility and prodigiosin production in Serratia

PubMed Central

Paterson, Thomas J.; Ney, Blair; Williamson, Neil R.; Easingwood, Richard A.; Bostina, Mihnea; Salmond, George P. C.

2016-01-01

SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon. PMID:27010574

Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression.

PubMed

Borirak, Orawan; Rolfe, Matthew D; de Koning, Leo J; Hoefsloot, Huub C J; Bekker, Martijn; Dekker, Henk L; Roseboom, Winfried; Green, Jeffrey; de Koster, Chris G; Hellingwerf, Klaas J

2015-10-01

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

The Ski oncoprotein interacts with the Smad proteins to repress TGFbeta signaling.

PubMed

Luo, K; Stroschein, S L; Wang, W; Chen, D; Martens, E; Zhou, S; Zhou, Q

1999-09-01

Smad proteins are critical signal transducers downstream of the receptors of the transforming growth factor-beta (TGFbeta) superfamily. On phosphorylation and activation by the active TGFbeta receptor complex, Smad2 and Smad3 form hetero-oligomers with Smad4 and translocate into the nucleus, where they interact with different cellular partners, bind to DNA, regulate transcription of various downstream response genes, and cross-talk with other signaling pathways. Here we show that a nuclear oncoprotein, Ski, can interact directly with Smad2, Smad3, and Smad4 on a TGFbeta-responsive promoter element and repress their abilities to activate transcription through recruitment of the nuclear transcriptional corepressor N-CoR and possibly its associated histone deacetylase complex. Overexpression of Ski in a TGFbeta-responsive cell line renders it resistant to TGFbeta-induced growth inhibition and defective in activation of JunB expression. This ability to overcome TGFbeta-induced growth arrest may be responsible for the transforming activity of Ski in human and avian cancer cells. Our studies suggest a new paradigm for inactivation of the Smad proteins by an oncoprotein through transcriptional repression.

The Ski oncoprotein interacts with the Smad proteins to repress TGFβ signaling

PubMed Central

Luo, Kunxin; Stroschein, Shannon L.; Wang, Wei; Chen, Dan; Martens, Eric; Zhou, Sharleen; Zhou, Qiang

1999-01-01

Smad proteins are critical signal transducers downstream of the receptors of the transforming growth factor-β (TGFβ) superfamily. On phosphorylation and activation by the active TGFβ receptor complex, Smad2 and Smad3 form hetero-oligomers with Smad4 and translocate into the nucleus, where they interact with different cellular partners, bind to DNA, regulate transcription of various downstream response genes, and cross-talk with other signaling pathways. Here we show that a nuclear oncoprotein, Ski, can interact directly with Smad2, Smad3, and Smad4 on a TGFβ-responsive promoter element and repress their abilities to activate transcription through recruitment of the nuclear transcriptional corepressor N-CoR and possibly its associated histone deacetylase complex. Overexpression of Ski in a TGFβ-responsive cell line renders it resistant to TGFβ-induced growth inhibition and defective in activation of JunB expression. This ability to overcome TGFβ-induced growth arrest may be responsible for the transforming activity of Ski in human and avian cancer cells. Our studies suggest a new paradigm for inactivation of the Smad proteins by an oncoprotein through transcriptional repression. PMID:10485843

The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT.

PubMed

Sun, Yi-Cheng; Guo, Xiao-Peng; Hinnebusch, B Joseph; Darby, Creg

2012-04-01

Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT.

Light represses transcription of asparagine synthetase genes in photosynthetic and nonphotosynthetic organs of plants.

PubMed Central

Tsai, F Y; Coruzzi, G

1991-01-01

Asparagine synthetase (AS) mRNA in Pisum sativum accumulates preferentially in plants grown in the dark. Nuclear run-on experiments demonstrate that expression of both the AS1 and AS2 genes is negatively regulated by light at the level of transcription. A decrease in the transcriptional rate of the AS1 gene can be detected as early as 20 min after exposure to light. Time course experiments reveal that the levels of AS mRNA fluctuate dramatically during a "normal" light/dark cycle. This is due to a direct effect of light and not to changes associated with circadian rhythm. A novel finding is that the light-repressed expression of the AS1 gene is as dramatic in nonphotosynthetic organs such as roots as it is in leaves. Experiments demonstrate that the small amount of light which passes through the soil is sufficient to repress AS1 expression in roots, indicating that light has a direct effect on AS1 gene expression in roots. The negative regulation of AS gene expression by light was shown to be a general phenomenon in plants which also occurs in nonlegumes such as Nicotiana plumbaginifolia and Nicotiana tabacum. Thus, the AS genes can serve as a model with which to dissect the molecular basis for light-regulated transcriptional repression in plants. Images PMID:1681424

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

PubMed

Bergmann, Jan H; Jakubsche, Julia N; Martins, Nuno M; Kagansky, Alexander; Nakano, Megumi; Kimura, Hiroshi; Kelly, David A; Turner, Bryan M; Masumoto, Hiroshi; Larionov, Vladimir; Earnshaw, William C

2012-01-15

Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity--kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

A noncanonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia.

PubMed

Shanmugam, Rajasubramaniam; Gade, Padmaja; Wilson-Weekes, Annique; Sayar, Hamid; Suvannasankha, Attaya; Goswami, Chirayu; Li, Lang; Gupta, Sushil; Cardoso, Angelo A; Baghdadi, Tareq Al; Sargent, Katie J; Cripe, Larry D; Kalvakolanu, Dhananjaya V; Boswell, H Scott

2012-01-15

Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB-and c-Jun-responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD(+) cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD(+) AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD(+) human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB-inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD(+) AMLs had selective nuclear activation of p52NF-κB. Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD(+) AML. ©2011 AACR.

A non-canonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia (AML)

PubMed Central

Shanmugam, Rajasubramaniam; Sayar, Hamid; Suvannasankha, Attaya; Goswami, Chirayu; Li, Lang; Gupta, Sushil; Cardoso, Angelo A.; Baghdadi, Tareq Al; Sargent, Katie J.; Cripe, Larry D.; Kalvakolanu, Dhananjaya V.; Boswell, H. Scott

2014-01-01

Purpose DAPK1, a tumor suppressor, is a rate-limiting effector in an ER stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of AML with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB- and c- jun-responsive genes, was studied. RNAi knockdown studies were performed in Flt3ITD+ve cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were performed to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. Results AMLs characterized by normal karyotype with Flt3ITD were found to have 10-100-fold lower DAPK1 transcripts normalized to the expression of c-jun, a transcriptional activator of DAPK1, as compared to a heterogeneous cytogenetic category. Meis1, a c-jun-responsive adverse AML prognostic gene signature was also measured as control. These Flt3ITD+ve AMLs over-express relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter along with HDAC2 and HDAC6 in the Flt3ITD+ve human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NIK, de-repressed DAPK1. DAPK1-repressed primary Flt3ITD+ve AMLs had selective nuclear activation of p52NF-κB. Conclusions Flt3ITD promotes a non-canonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD+ve AML. PMID:22096027

Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle

PubMed Central

Khurana, Simran; Warburton, Alix

2017-01-01

We have shown previously that Sp100 (a component of the ND10 nuclear body) represses transcription, replication and establishment of incoming human papillomavirus (HPV) DNA in the early stages of infection. In this follow up study, we show that Sp100 does not substantially regulate viral infection in the maintenance phase, however at late stages of infection Sp100 interacts with amplifying viral genomes to repress viral processes. We find that Sp100 localizes to HPV16 replication foci generated in primary keratinocytes, to HPV31 replication foci that form in differentiated cells, and to HPV16 replication foci in CIN 1 cervical biopsies. To analyze this further, Sp100 was down regulated by siRNA treatment of differentiating HPV31 containing cells and levels of viral transcription and replication were assessed. This revealed that Sp100 represses viral transcription and replication in differentiated cells. Analysis of Sp100 binding to viral chromatin showed that Sp100 bound across the viral genome, and that binding increased at late stages of infection. Therefore, Sp100 represses the HPV life cycle at both early and late stages of infection. PMID:28968443

A gene regulatory network armature for T-lymphocyte specification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fung, Elizabeth-sharon

Choice of a T-lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly-regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification; tests of the short-term Notchdependence of these gene expression changes; and perturbation analyses of the effects of overexpression of two essential transcription factors, namely PU.l and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through whichmore » T-cell precursors progress from primitive multipotency to T-lineage commitment. Distinct parts of the path reveal separate contributions of Notch signaling, GATA-3 activity, and downregulation of PU.l. Using BioTapestry, the results have been assembled into a draft gene regulatory network for the specification of T-cell precursors and the choice of T as opposed to myeloid dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfil against Egr-2 and of TCF-l against PU.l as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose-dependence of GATA-3 effects; the gene-specific modulation of PU.l activity based on Notch activity; the lack of direct opposition between PU.l and GATA-3; and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.« less

Regulation of MET by FOXP2, genes implicated in higher cognitive dysfunction and autism risk.

PubMed

Mukamel, Zohar; Konopka, Genevieve; Wexler, Eric; Osborn, Gregory E; Dong, Hongmei; Bergman, Mica Y; Levitt, Pat; Geschwind, Daniel H

2011-08-10

Autism spectrum disorder (ASD) is a highly heritable, behaviorally defined, heterogeneous disorder of unknown pathogenesis. Several genetic risk genes have been identified, including the gene encoding the receptor tyrosine kinase MET, which regulates neuronal differentiation and growth. An ASD-associated polymorphism disrupts MET gene transcription, and there are reduced levels of MET protein expression in the mature temporal cortex of subjects with ASD. To address the possible neurodevelopmental contribution of MET to ASD pathogenesis, we examined the expression and transcriptional regulation of MET by a transcription factor, FOXP2, which is implicated in regulation of cognition and language, two functions altered in ASD. MET mRNA expression in the midgestation human fetal cerebral cortex is strikingly restricted, localized to portions of the temporal and occipital lobes. Within the cortical plate of the temporal lobe, the pattern of MET expression is highly complementary to the expression pattern of FOXP2, suggesting the latter may play a role in repression of gene expression. Consistent with this, MET and FOXP2 also are reciprocally expressed by differentiating normal human neuronal progenitor cells (NHNPs) in vitro, leading us to assess whether FOXP2 transcriptionally regulates MET. Indeed, FOXP2 binds directly to the 5' regulatory region of MET, and overexpression of FOXP2 results in transcriptional repression of MET. The expression of MET in restricted human neocortical regions, and its regulation in part by FOXP2, is consistent with genetic evidence for MET contributing to ASD risk.

MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

PubMed Central

Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

2016-01-01

MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

Phosphorylation of Trihelix Transcriptional Repressor ASR3 by MAP KINASE4 Negatively Regulates Arabidopsis Immunity

PubMed Central

Li, Bo; Jiang, Shan; Yu, Xiao; Cheng, Cheng; Chen, Sixue; Cheng, Yanbing; Yuan, Joshua S.; Jiang, Daohong; He, Ping; Shan, Libo

2015-01-01

Proper control of immune-related gene expression is crucial for the host to launch an effective defense response. Perception of microbe-associated molecular patterns (MAMPs) induces rapid and profound transcriptional reprogramming via unclear mechanisms. Here, we show that ASR3 (ARABIDOPSIS SH4-RELATED3) functions as a transcriptional repressor and plays a negative role in regulating pattern-triggered immunity (PTI) in Arabidopsis thaliana. ASR3 belongs to a plant-specific trihelix transcription factor family for which functional studies are lacking. MAMP treatments induce rapid phosphorylation of ASR3 at threonine 189 via MPK4, a mitogen-activated protein kinase that negatively regulates PTI responses downstream of multiple MAMP receptors. ASR3 possesses transcriptional repressor activity via its ERF-associated amphiphilic repression motifs and negatively regulates a large subset of flg22-induced genes. Phosphorylation of ASR3 by MPK4 enhances its DNA binding activity to suppress gene expression. Importantly, the asr3 mutant shows enhanced disease resistance to virulent bacterial pathogen infection, whereas transgenic plants overexpressing the wild-type or phospho-mimetic form of ASR3 exhibit compromised PTI responses. Our studies reveal a function of the trihelix transcription factors in plant innate immunity and provide evidence that ASR3 functions as a transcriptional repressor regulated by MAMP-activated MPK4 to fine-tune plant immune gene expression. PMID:25770109

Cerberus-Nodal-Lefty-Pitx signaling cascade controls left-right asymmetry in amphioxus.

PubMed

Li, Guang; Liu, Xian; Xing, Chaofan; Zhang, Huayang; Shimeld, Sebastian M; Wang, Yiquan

2017-04-04

Many bilaterally symmetrical animals develop genetically programmed left-right asymmetries. In vertebrates, this process is under the control of Nodal signaling, which is restricted to the left side by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest diverging chordate lineage, has profound left-right asymmetry as a larva. We show that Cerberus , Nodal , Lefty , and their target transcription factor Pitx are sequentially activated in amphioxus embryos. We then address their function by transcription activator-like effector nucleases (TALEN)-based knockout and heat-shock promoter (HSP)-driven overexpression. Knockout of Cerberus leads to ectopic right-sided expression of Nodal , Lefty , and Pitx , whereas overexpression of Cerberus represses their left-sided expression. Overexpression of Nodal in turn represses Cerberus and activates Lefty and Pitx ectopically on the right side. We also show Lefty represses Nodal , whereas Pitx activates Nodal These data combine in a model in which Cerberus determines whether the left-sided gene expression cassette is activated or repressed. These regulatory steps are essential for normal left-right asymmetry to develop, as when they are disrupted embryos may instead form two phenotypic left sides or two phenotypic right sides. Our study shows the regulatory cassette controlling left-right asymmetry was in place in the ancestor of amphioxus and vertebrates. This includes the Nodal inhibitors Cerberus and Lefty, both of which operate in feedback loops with Nodal and combine to establish asymmetric Pitx expression. Cerberus and Lefty are missing from most invertebrate lineages, marking this mechanism as an innovation in the lineage leading to modern chordates.

Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.

PubMed

Smith, Justin D; Suresh, Sundari; Schlecht, Ulrich; Wu, Manhong; Wagih, Omar; Peltz, Gary; Davis, Ronald W; Steinmetz, Lars M; Parts, Leopold; St Onge, Robert P

2016-03-08

Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae. We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets. Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.

Cul3-mediated Nrf2 ubiquitination and antioxidant response element (ARE) activation are dependent on the partial molar volume at position 151 of Keap1.

PubMed

Eggler, Aimee L; Small, Evan; Hannink, Mark; Mesecar, Andrew D

2009-07-29

Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that activates transcription of a battery of cytoprotective genes by binding to the ARE (antioxidant response element). Nrf2 is repressed by the cysteine-rich Keap1 (kelch-like ECH-associated protein 1) protein, which targets Nrf2 for ubiquitination and subsequent degradation by a Cul3 (cullin 3)-mediated ubiquitination complex. We find that modification of Cys(151) of human Keap1, by mutation to a tryptophan, relieves the repression by Keap1 and allows activation of the ARE by Nrf2. The Keap1 C151W substitution has a decreased affinity for Cul3, and can no longer serve to target Nrf2 for ubiquitination, though it retains its affinity for Nrf2. A series of 12 mutant Keap1 proteins, each containing a different residue at position 151, was constructed to explore the chemistry required for this effect. The series reveals that the extent to which Keap1 loses the ability to target Nrf2 for degradation, and hence the ability to repress ARE activation, correlates well with the partial molar volume of the residue. Other physico-chemical properties do not appear to contribute significantly to the effect. Based on this finding, a structural model is proposed whereby large residues at position 151 cause steric clashes that lead to alteration of the Keap1-Cul3 interaction. This model has significant implications for how electrophiles which modify Cys(151), disrupt the repressive function of Keap1.

IL-1β-specific recruitment of GCN5 histone acetyltransferase induces the release of PAF1 from chromatin for the de-repression of inflammatory response genes.

PubMed

Kim, Nari; Sun, Hwa-Young; Youn, Min-Young; Yoo, Joo-Yeon

2013-04-01

To determine the functional specificity of inflammation, it is critical to orchestrate the timely activation and repression of inflammatory responses. Here, we explored the PAF1 (RNA polymerase II associated factor)-mediated signal- and locus-specific repression of genes induced through the pro-inflammatory cytokine interleukin (IL)-1β. Using microarray analysis, we identified the PAF1 target genes whose expression was further enhanced by PAF1 knockdown in IL-1β-stimulated HepG2 hepatocarcinomas. PAF1 bound near the transcription start sites of target genes and dissociated on stimulation. In PAF1-deficient cells, more elongating RNA polymerase II and acetylated histones were observed, although IL-1β-mediated activation and recruitment of nuclear factor κB (NF-κB) were not altered. Under basal conditions, PAF1 blocked histone acetyltransferase general control non-depressible 5 (GCN5)-mediated acetylation on H3K9 and H4K5 residues. On IL-1β stimulation, activated GCN5 discharged PAF1 from chromatin, allowing productive transcription to occur. PAF1 bound to histones but not to acetylated histones, and the chromatin-binding domain of PAF1 was essential for target gene repression. Moreover, IL-1β-induced cell migration was similarly controlled through counteraction between PAF1 and GCN5. These results suggest that the IL-1β signal-specific exchange of PAF1 and GCN5 on the target locus limits inappropriate gene induction and facilitates the timely activation of inflammatory responses.

rahu is a mutant allele of Dnmt3c, encoding a DNA methyltransferase homolog required for meiosis and transposon repression in the mouse male germline

PubMed Central

Lange, Julian; Lailler, Nathalie

2017-01-01

Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase paralogs—Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l—that establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called ‘rahu’, which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation. PMID:28854222

CcpA Ensures Optimal Metabolic Fitness of Streptococcus pneumoniae

PubMed Central

Kuipers, Oscar P.; Neves, Ana Rute

2011-01-01

In Gram-positive bacteria, the transcriptional regulator CcpA is at the core of catabolite control mechanisms. In the human pathogen Streptococcus pneumoniae, links between CcpA and virulence have been established, but its role as a master regulator in different nutritional environments remains to be elucidated. Thus, we performed whole-transcriptome and metabolic analyses of S. pneumoniae D39 and its isogenic ccpA mutant during growth on glucose or galactose, rapidly and slowly metabolized carbohydrates presumably encountered by the bacterium in different host niches. CcpA affected the expression of up to 19% of the genome covering multiple cellular processes, including virulence, regulatory networks and central metabolism. Its prevalent function as a repressor was observed on glucose, but unexpectedly also on galactose. Carbohydrate-dependent CcpA regulation was also observed, as for the tagatose 6-phosphate pathway genes, which were activated by galactose and repressed by glucose. Metabolite analyses revealed that two pathways for galactose catabolism are functionally active, despite repression of the Leloir genes by CcpA. Surprisingly, galactose-induced mixed-acid fermentation apparently required CcpA, since genes involved in this type of metabolism were mostly under CcpA-repression. These findings indicate that the role of CcpA extends beyond transcriptional regulation, which seemingly is overlaid by other regulatory mechanisms. In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation. Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels. Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall. Hence, CcpA may play a key role in mediating the interaction of S. pneumoniae with its host. Overall, our results support the hypothesis that S. pneumoniae optimizes basic metabolic processes, likely enhancing in vivo fitness, in a CcpA-mediated manner. PMID:22039538

Functional interrogation of non-coding DNA through CRISPR genome editing.

PubMed

Canver, Matthew C; Bauer, Daniel E; Orkin, Stuart H

2017-05-15

Methodologies to interrogate non-coding regions have lagged behind coding regions despite comprising the vast majority of the genome. However, the rapid evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing has provided a multitude of novel techniques for laboratory investigation including significant contributions to the toolbox for studying non-coding DNA. CRISPR-mediated loss-of-function strategies rely on direct disruption of the underlying sequence or repression of transcription without modifying the targeted DNA sequence. CRISPR-mediated gain-of-function approaches similarly benefit from methods to alter the targeted sequence through integration of customized sequence into the genome as well as methods to activate transcription. Here we review CRISPR-based loss- and gain-of-function techniques for the interrogation of non-coding DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

DDM1 represses noncoding RNA expression and RNA-directed DNA methylation in heterochromatin.

PubMed

Tan, Feng; Lu, Yue; Jiang, Wei; Zhao, Yu; Wu, Tian; Zhang, Ruoyu; Zhou, Dao-Xiu

2018-05-24

Cytosine methylation of DNA, which occurs at CG, CHG, and CHH (H=A, C, or T) sequences in plants, is a hallmark for epigenetic repression of repetitive sequences. The chromatin remodeling factor DECREASE IN DNA METHYLATION1 (DDM1) is essential for DNA methylation, especially at CG and CHG sequences. However, its potential role in RNA-directed DNA methylation (RdDM) and in chromatin function is not completely understood in rice (Oryza sativa). In this work, we used high-throughput approaches to study the function of rice DDM1 (OsDDM1) in RdDM and the expression of non-coding RNA (ncRNA). We show that loss of function of OsDDM1 results in ectopic CHH methylation of transposable elements and repeats. The ectopic CHH methylation was dependent on rice DOMAINS REARRANGED METHYLTRANSFERASE2 (OsDRM2), a DNA methyltransferase involved in RdDM. Mutations in OsDDM1 lead to decreases of histone H3K9me2 and increases in the levels of heterochromatic small RNA (sRNA) and long noncoding RNA (lncRNA). In particular, OsDDM1 was found to be essential to repress transcription of the two repetitive sequences, Centromeric Retrotransposons of Rice1 (CRR1) and the dominant centromeric CentO repeats. These results suggest that OsDDM1 antagonizes RdDM at heterochromatin and represses tissue-specific expression of ncRNA from repetitive sequences in the rice genome. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Orphan nuclear receptor TLX recruits histone deacetylases to repress transcription and regulate neural stem cell proliferation.

PubMed

Sun, Guoqiang; Yu, Ruth T; Evans, Ronald M; Shi, Yanhong

2007-09-25

TLX is a transcription factor that is essential for neural stem cell proliferation and self-renewal. However, the molecular mechanism of TLX-mediated neural stem cell proliferation and self-renewal is largely unknown. We show here that TLX recruits histone deacetylases (HDACs) to its downstream target genes to repress their transcription, which in turn regulates neural stem cell proliferation. TLX interacts with HDAC3 and HDAC5 in neural stem cells. The HDAC5-interaction domain was mapped to TLX residues 359-385, which contains a conserved nuclear receptor-coregulator interaction motif IXXLL. Both HDAC3 and HDAC5 have been shown to be recruited to the promoters of TLX target genes along with TLX in neural stem cells. Recruitment of HDACs led to transcriptional repression of TLX target genes, the cyclin-dependent kinase inhibitor, p21(CIP1/WAF1)(p21), and the tumor suppressor gene, pten. Either inhibition of HDAC activity or knockdown of HDAC expression led to marked induction of p21 and pten gene expression and dramatically reduced neural stem cell proliferation, suggesting that the TLX-interacting HDACs play an important role in neural stem cell proliferation. Moreover, expression of a TLX peptide containing the minimal HDAC5 interaction domain disrupted the TLX-HDAC5 interaction. Disruption of this interaction led to significant induction of p21 and pten gene expression and to dramatic inhibition of neural stem cell proliferation. Taken together, these findings demonstrate a mechanism for neural stem cell proliferation through transcriptional repression of p21 and pten gene expression by TLX-HDAC interactions.

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene

PubMed Central

Kim, Seongman; Dai, Gan; O’Callaghan, Dennis J.; Kim, Seong Kee

2012-01-01

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5’-ATCGT-3’) located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box. PMID:22265772

Characterization of cis-acting elements required for autorepression of the equine herpesvirus 1 IE gene.

PubMed

Kim, Seongman; Dai, Gan; O'Callaghan, Dennis J; Kim, Seong Kee

2012-04-01

The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5'-ATCGT-3') located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box. Copyright © 2012. Published by Elsevier B.V.

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

PubMed Central

Baribault, Carl; Ehrlich, Kenneth C.; Ponnaluri, V. K. Chaithanya; Pradhan, Sriharsa; Lacey, Michelle; Ehrlich, Melanie

2018-01-01

ABSTRACT DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development. PMID:29498561

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

PubMed

Garvin, Lindsay M; Chen, Yajun; Damsker, Jesse M; Rose, Mary C

2016-06-01

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1β upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1β in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nuclear Receptor SHP Activates miR-206 Expression via a Cascade Dual Inhibitory Mechanism

PubMed Central

Song, Guisheng; Wang, Li

2009-01-01

MicroRNAs play a critical role in many essential cellular functions in the mammalian species. However, limited information is available regarding the regulation of miRNAs gene transcription. Microarray profiling and real-time PCR analysis revealed a marked down-regulation of miR-206 in nuclear receptor SHP−/− mice. To understand the regulatory function of SHP with regard to miR-206 gene expression, we determined the putative transcriptional initiation site of miR-206 and also its full length primary transcript using a database mining approach and RACE. We identified the transcription factor AP1 binding sites on the miR-206 promoter and further showed that AP1 (c-Jun and c-Fos) induced miR-206 promoter transactivity and expression which was repressed by YY1. ChIP analysis confirmed the physical association of AP1 (c-Jun) and YY1 with the endogenous miR-206 promoter. In addition, we also identified nuclear receptor ERRγ (NR3B3) binding site on the YY1 promoter and showed that YY1 promoter was transactivated by ERRγ, which was inhibited by SHP (NROB2). ChIP analysis confirmed the ERRγ binding to the YY1 promoter. Forced expression of SHP and AP1 induced miR-206 expression while overexpression of ERRγ and YY1 reduced its expression. The effects of AP1, ERRγ, and YY1 on miR-206 expression were reversed by siRNA knockdown of each gene, respectively. Thus, we propose a novel cascade “dual inhibitory” mechanism governing miR-206 gene transcription by SHP: SHP inhibition of ERRγ led to decreased YY1 expression and the de-repression of YY1 on AP1 activity, ultimately leading to the activation of miR-206. This is the first report to elucidate a cascade regulatory mechanism governing miRNAs gene transcription. PMID:19721712

Epigenetic control of skull morphogenesis by histone deacetylase 8

PubMed Central

Haberland, Michael; Mokalled, Mayssa H.; Montgomery, Rusty L.; Olson, Eric N.

2009-01-01

Histone deacetylases (Hdacs) are transcriptional repressors with crucial roles in mammalian development. Here we provide evidence that Hdac8 specifically controls patterning of the skull by repressing a subset of transcription factors in cranial neural crest cells. Global deletion of Hdac8 in mice leads to perinatal lethality due to skull instability, and this is phenocopied by conditional deletion of Hdac8 in cranial neural crest cells. Hdac8 specifically represses the aberrant expression of homeobox transcription factors such as Otx2 and Lhx1. These findings reveal how the identity and patterning of vertebrate-specific portions of the skull are epigenetically controlled by a histone deacetylase. PMID:19605684

Nuclear Function of Smad7 Promotes Myogenesis▿

PubMed Central

Miyake, Tetsuaki; Alli, Nezeka S.; McDermott, John C.

2010-01-01

In the “canonical” view of transforming growth factor β (TGF-β) signaling, Smad7 plays an inhibitory role. While Smad7 represses Smad3 activation by TGF-β, it does not reverse the inhibitory effect of TGF-β on myogenesis, suggesting a different function in myogenic cells. We previously reported a promyogenic role of Smad7 mediated by an interaction with MyoD. Based on this association, we hypothesized a possible nuclear function of Smad7 independent of its role at the level of the receptor. We therefore engineered a chimera of Smad7 with a nuclear localization signal (NLS), which serves to prevent and therefore bypass binding to the TGF-β receptor while concomitantly constitutively localizing Smad7 to the nucleus. This Smad7-NLS did not repress Smad3 activation by TGF-β but did retain its ability to enhance myogenic gene activation and phenotypic myogenesis, indicating that the nuclear, receptor-independent function of Smad7 is sufficient to promote myogenesis. Furthermore, Smad7 physically interacts with MyoD and antagonizes the repressive effects of active MEK on MyoD. Reporter and myogenic conversion assays indicate a pivotal regulation of MyoD transcriptional properties by the balance between Smad7 and active MEK. Thus, Smad7 has a nuclear coactivator function that is independent of TGF-β signaling and necessary to promote myogenic differentiation. PMID:19995910

Specialized sugar sensing in diverse fungi.

PubMed

Brown, Victoria; Sabina, Jeffrey; Johnston, Mark

2009-03-10

S. cerevisiae senses glucose and galactose differently. Glucose is detected through sensors that reside in the cellular plasma membrane. When activated, the sensors initiate a signal-transduction cascade that ultimately inactivates the Rgt1 transcriptional repressor by causing degradation of its corepressors Mth1 and Std1. This results in the expression of many HXT genes encoding glucose transporters. The ensuing flood of glucose into the cell activates Mig1, a transcriptional repressor that mediates "glucose repression" of many genes, including the GAL genes; hence, glucose sensing hinders galactose utilization. Galactose is sensed in the cytoplasm via Gal3. Upon binding galactose (and ATP), Gal3 sequesters the Gal80 protein, thereby emancipating the Gal4 transcriptional activator of the GAL genes. Gal4 also activates expression of MTH1, encoding a corepressor critical for Rgt1 function. Thus, galactose inhibits glucose assimilation by encouraging repression of HXT genes. C. albicans senses glucose similarly to S. cerevisiae but does not sense galactose through Gal3-Gal80-Gal4. Its genome harbors no GAL80 ortholog, and the severely truncated CaGal4 does not regulate CaGAL genes. We present evidence that C. albicans senses galactose with its Hgt4 glucose sensor, a capability that is enabled by transcriptional "rewiring" of its sugar-sensing signal-transduction pathways. We suggest that galactose sensing through Hgt4 is ancestral in fungi.

Arabidopsis WRKY6 Transcription Factor Acts as a Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development

PubMed Central

Wu, Wei-Hua; Chen, Yi-Fang

2016-01-01

The phytohormone abscisic acid (ABA) plays important roles during seed germination and early seedling development. Here, we characterized the function of the Arabidopsis WRKY6 transcription factor in ABA signaling. The transcript of WRKY6 was repressed during seed germination and early seedling development, and induced by exogenous ABA. The wrky6-1 and wrky6-2 mutants were ABA insensitive, whereas WRKY6-overexpressing lines showed ABA-hypersensitive phenotypes during seed germination and early seedling development. The expression of RAV1 was suppressed in the WRKY6-overexpressing lines and elevated in the wrky6 mutants, and the expression of ABI3, ABI4, and ABI5, which was directly down-regulated by RAV1, was enhanced in the WRKY6-overexpressing lines and repressed in the wrky6 mutants. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that WRKY6 could bind to the RAV1 promoter in vitro and in vivo. Overexpression of RAV1 in WRKY6-overexpressing lines abolished their ABA-hypersensitive phenotypes, and the rav1 wrky6-2 double mutant showed an ABA-hypersensitive phenotype, similar to rav1 mutant. Together, the results demonstrated that the Arabidopsis WRKY6 transcription factor played important roles in ABA signaling by directly down-regulating RAV1 expression. PMID:26829043

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

PubMed Central

Avram, Dorina; Fields, Andrew; Senawong, Thanaset; Topark-Ngarm, Acharawan; Leid, Mark

2002-01-01

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems. PMID:12196208

Post transcriptional control of the epigenetic stem cell regulator PLZF by sirtuin and HDAC deacetylases.

PubMed

McConnell, Melanie J; Durand, Laetitia; Langley, Emma; Coste-Sarguet, Lise; Zelent, Arthur; Chomienne, Christine; Kouzarides, Tony; Licht, Jonathan D; Guidez, Fabien

2015-01-01

The transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) is critical for the regulation of normal stem cells maintenance by establishing specific epigenetic landscape. We have previously shown that CBP/p300 acetyltransferase induces PLZF acetylation in order to increase its deoxynucleotidic acid (DNA) binding activity and to enhance its epigenetic function (repression of PLZF target genes). However, how PLZF is inactivated is not yet understood. In this study, we demonstrate that PLZF is deacetylated by both histone deacetylase 3 and the NAD+ dependent deacetylase silent mating type information regulation 2 homolog 1 (SIRT1). Unlike other PLZF-interacting deacetylases, these two proteins interact with the zinc finger domain of PLZF, where the activating CBP/p300 acetylation site was previously described, inducing deacetylation of lysines 647/650/653. Overexpression of histone deacetylase 3 (HDAC3) and SIRT1 is associated with loss of PLZF DNA binding activity and decreases PLZF transcriptional repression. As a result, the chromatin status of the promoters of PLZF target genes, involved in oncogenesis, shift from a heterochromatin to an open euchromatin environment leading to gene expression even in the presence of PLZF. Consequently, SIRT1 and HDAC3 mediated-PLZF deacetylation provides for rapid control and fine-tuning of PLZF activity through post-transcriptional modification to regulate gene expression and cellular homeostasis.

Role of Nuclear Lamina in Gene Repression and Maintenance of Chromosome Architecture in the Nucleus.

PubMed

Shevelyov, Y Y; Ulianov, S V

2018-04-01

Nuclear lamina is a protein meshwork composed of lamins and lamin-associated proteins that lines the nuclear envelope from the inside and forms repressive transcription compartment. The review presents current data on the contribution of nuclear lamina to the repression of genes located in this compartment and on the mechanisms of chromatin attachment to the nuclear envelope.

PIE-1 is a bifunctional protein that regulates maternal and zygotic gene expression in the embryonic germ line of Caenorhabditis elegans

PubMed Central

Tenenhaus, Christina; Subramaniam, Kuppuswamy; Dunn, Melanie A.; Seydoux, Geraldine

2001-01-01

The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage during the first cleavages of the Caenorhabditis elegans embryo. We have shown previously that one function of PIE-1 is to inhibit mRNA transcription. Here we show that PIE-1 has a second function in germ cells; it is required for efficient expression of the maternally encoded Nanos homolog NOS-2. This second function is genetically separable from PIE-1's inhibitory effect on transcription. A mutation in PIE-1's second CCCH finger reduces NOS-2 expression without affecting transcriptional repression and causes primordial germ cells to stray away from the somatic gonad, occasionally exiting the embryo entirely. Our results indicate that PIE-1 promotes germ cell fate by two independent mechanisms as follows: (1) inhibition of transcription, which blocks zygotic programs that drive somatic development, and (2) activation of protein expression from nos-2 and possibly other maternal RNAs, which promotes primordial germ cell development. PMID:11316796

Mlc Is a Transcriptional Activator with a Key Role in Integrating Cyclic AMP Receptor Protein and Integration Host Factor Regulation of Leukotoxin RNA Synthesis in Aggregatibacter actinomycetemcomitans

PubMed Central

Childress, Catherine; Feuerbacher, Leigh A.; Phillips, Linda; Burgum, Alex

2013-01-01

Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cyclic AMP (cAMP) receptor protein (CRP) indirectly increases ltxA expression, but the intermediary regulator is unknown. Integration host factor (IHF) binds to and represses the leukotoxin promoter, but neither CRP nor IHF is responsible for the anaerobic induction of ltxA RNA synthesis. Thus, we have undertaken studies to identify other regulators of leukotoxin transcription and to demonstrate how these proteins work together to modulate leukotoxin synthesis. First, analyses of ltxA RNA expression from defined leukotoxin promoter mutations in the chromosome identify positions −69 to −35 as the key control region and indicate that an activator protein modulates leukotoxin transcription. We show that Mlc, which is a repressor in Escherichia coli, functions as a direct transcriptional activator in A. actinomycetemcomitans; an mlc deletion mutant reduces leukotoxin RNA synthesis, and recombinant Mlc protein binds specifically at the −68 to −40 region of the leukotoxin promoter. Furthermore, we show that CRP activates ltxA expression indirectly by increasing the levels of Mlc. Analyses of Δmlc, Δihf, and Δihf Δmlc strains demonstrate that Mlc can increase RNA polymerase (RNAP) activity directly and that IHF represses ltxA RNA synthesis mainly by blocking Mlc binding. Finally, a Δihf Δmlc mutant still induces ltxA during anaerobic growth, indicating that there are additional factors involved in leukotoxin transcriptional regulation. A model for the coordinated regulation of leukotoxin transcription is presented. PMID:23475968

Hijacking T cell differentiation: new insights in TLX function in T-ALL.

PubMed

King, Bryan; Ntziachristos, Panagiotis; Aifantis, Iannis

2012-04-17

TLX1 and TLX3 are two closely-related homeobox transcriptional repressors frequently misexpressed and translocated in T cell acute lymphoblastic leukemia (T-ALL). In this issue of Cancer Cell, Dadi et al. provide new insights into how these factors are recruited by ETS-1 to the TCRα enhancer and actively repress differentiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Transcription Factor IRF4 Promotes CD8+ T Cell Exhaustion and Limits the Development of Memory-like T Cells during Chronic Infection.

PubMed

Man, Kevin; Gabriel, Sarah S; Liao, Yang; Gloury, Renee; Preston, Simon; Henstridge, Darren C; Pellegrini, Marc; Zehn, Dietmar; Berberich-Siebelt, Friederike; Febbraio, Mark A; Shi, Wei; Kallies, Axel

2017-12-19

During chronic stimulation, CD8 + T cells acquire an exhausted phenotype characterized by expression of inhibitory receptors, down-modulation of effector function, and metabolic impairments. T cell exhaustion protects from excessive immunopathology but limits clearance of virus-infected or tumor cells. We transcriptionally profiled antigen-specific T cells from mice infected with lymphocytic choriomeningitis virus strains that cause acute or chronic disease. T cell exhaustion during chronic infection was driven by high amounts of T cell receptor (TCR)-induced transcription factors IRF4, BATF, and NFATc1. These regulators promoted expression of inhibitory receptors, including PD-1, and mediated impaired cellular metabolism. Furthermore, they repressed the expression of TCF1, a transcription factor required for memory T cell differentiation. Reducing IRF4 expression restored the functional and metabolic properties of antigen-specific T cells and promoted memory-like T cell development. These findings indicate that IRF4 functions as a central node in a TCR-responsive transcriptional circuit that establishes and sustains T cell exhaustion during chronic infection. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Adaptor proteins GIR1 and GIR2. I. Interaction with the repressor GLABRA2 and regulation of root hair development.

PubMed

Wu, Renhong; Citovsky, Vitaly

2017-07-01

Plants use specialized root outgrowths, termed root hairs, to enhance acquisition of nutrients and water, help secure anchorage, and facilitate interactions with soil microbiome. One of the major regulators of this process is GLABRA2 (GL2), a transcriptional repressor of root hair differentiation. However, regulation of the GL2-function is relatively well characterized, it remains completely unknown whether GL2 itself functions in complex with other transcriptional regulators. We identified GIR1 and GIR2, a plant-specific two-member family of closely related proteins that interact with GL2. Loss-of-function mutants of GIR1 and GIR2 enhanced development of root hair whereas gain-of-function mutants repressed it. Thus, GIR1 and GIR2 might function as adaptor proteins that associate with GL2 and participate in control of root hair formation. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of cytosine methylation on transcription factor binding sites

PubMed Central

2014-01-01

Background DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect the affinity of transcription factors (TFs) for their binding sites (TFBSs). If it is a consequence, then gene repression caused by chromatin modification may be stabilized by DNA methylation. Until now, these two possibilities have been supported only by non-systematic evidence and they have not been tested on a wide range of TFs. An average promoter methylation is usually used in studies, whereas recent results suggested that methylation of individual cytosines can also be important. Results We found that the methylation profiles of 16.6% of cytosines and the expression profiles of neighboring transcriptional start sites (TSSs) were significantly negatively correlated. We called the CpGs corresponding to such cytosines “traffic lights”. We observed a strong selection against CpG “traffic lights” within TFBSs. The negative selection was stronger for transcriptional repressors as compared with transcriptional activators or multifunctional TFs as well as for core TFBS positions as compared with flanking TFBS positions. Conclusions Our results indicate that direct and selective methylation of certain TFBS that prevents TF binding is restricted to special cases and cannot be considered as a general regulatory mechanism of transcription. PMID:24669864

The Transcriptional Coregulator LEUNIG_HOMOLOG Inhibits Light-Dependent Seed Germination in Arabidopsis

PubMed Central

Lee, Nayoung; Park, Jeongmoo; Kim, Keunhwa; Choi, Giltsu

2015-01-01

PHYTOCHROME-INTERACTING FACTOR1 (PIF1) is a basic helix-loop-helix transcription factor that inhibits light-dependent seed germination in Arabidopsis thaliana. However, it remains unclear whether PIF1 requires other factors to regulate its direct targets. Here, we demonstrate that LEUNIG_HOMOLOG (LUH), a Groucho family transcriptional corepressor, binds to PIF1 and coregulates its targets. Not only are the transcriptional profiles of the luh and pif1 mutants remarkably similar, more than 80% of the seeds of both genotypes germinate in the dark. We show by chromatin immunoprecipitation that LUH binds a subset of PIF1 targets in a partially PIF1-dependent manner. Unexpectedly, we found LUH binds and coregulates not only PIF1-activated targets but also PIF1-repressed targets. Together, our results indicate LUH functions with PIF1 as a transcriptional coregulator to inhibit seed germination. PMID:26276832

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less

Regulation of a maize HD-ZIP IV transcription factor by a non-conventional RDR2-dependent small RNA.

PubMed

Klein-Cosson, Catherine; Chambrier, Pierre; Rogowsky, Peter M; Vernoud, Vanessa

2015-03-01

Small non-coding RNAs are versatile riboregulators that control gene expression at the transcriptional or post-transcriptional level, governing many facets of plant development. Here we present evidence for the existence of a 24 nt small RNA (named small1) that is complementary to the 3' UTR of OCL1 (Outer Cell Layer1), the founding member of the maize HD-ZIP IV gene family encoding plant-specific transcription factors that are mainly involved in epidermis differentiation and specialization. The biogenesis of small1 depends on DICER-like 3 (DCL3), RNA-dependent RNA polymerase 2 (RDR2) and RNA polymerase IV, components that are usually required for RNA-dependent DNA-methylation. Unexpectedly, GFP sensor experiments in transient and stable transformation systems revealed that small1 may regulate its target at the post-transcriptional level, mainly through translational repression. This translational repression is attenuated in an rdr2 mutant background in which small1 does not accumulate. Our experiments further showed the possible involvement of a secondary stem-loop structure present in the 3' UTR of OCL1 for efficient target repression, suggesting the existence of several regulatory mechanisms affecting OCL1 mRNA stability and translation. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

GTL1 and DF1 regulate root hair growth through transcriptional repression of ROOT HAIR DEFECTIVE 6-LIKE 4 in Arabidopsis.

PubMed

Shibata, Michitaro; Breuer, Christian; Kawamura, Ayako; Clark, Natalie M; Rymen, Bart; Braidwood, Luke; Morohashi, Kengo; Busch, Wolfgang; Benfey, Philip N; Sozzani, Rosangela; Sugimoto, Keiko

2018-02-08

How plants determine the final size of growing cells is an important, yet unresolved, issue. Root hairs provide an excellent model system with which to study this as their final cell size is remarkably constant under constant environmental conditions. Previous studies have demonstrated that a basic helix-loop helix transcription factor ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promotes root hair growth, but how hair growth is terminated is not known. In this study, we demonstrate that a trihelix transcription factor GT-2-LIKE1 (GTL1) and its homolog DF1 repress root hair growth in Arabidopsis Our transcriptional data, combined with genome-wide chromatin-binding data, show that GTL1 and DF1 directly bind the RSL4 promoter and regulate its expression to repress root hair growth. Our data further show that GTL1 and RSL4 regulate each other, as well as a set of common downstream genes, many of which have previously been implicated in root hair growth. This study therefore uncovers a core regulatory module that fine-tunes the extent of root hair growth by the orchestrated actions of opposing transcription factors. © 2018. Published by The Company of Biologists Ltd.

Cation-induced transcriptional regulation of the dlt operon of Staphylococcus aureus.

PubMed

Koprivnjak, Tomaz; Mlakar, Vid; Swanson, Lindsey; Fournier, Benedicte; Peschel, Andreas; Weiss, Jerrold P

2006-05-01

Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with D-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. D-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating D-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na(+) and moderate concentrations of Mg(2+) and Ca(2+) but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg(2+)-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg(2+)-induced repression of the dlt operon in S. aureus.

Cation-Induced Transcriptional Regulation of the dlt Operon of Staphylococcus aureus

PubMed Central

Koprivnjak, Tomaz; Mlakar, Vid; Swanson, Lindsey; Fournier, Benedicte; Peschel, Andreas; Weiss, Jerrold P.

2006-01-01

Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with d-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. d-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating d-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na+ and moderate concentrations of Mg2+ and Ca2+ but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg2+-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg2+-induced repression of the dlt operon in S. aureus. PMID:16672616

The nuclear hormone receptor SEX-1 is an X-chromosome signal that determines nematode sex.

PubMed

Carmi, I; Kopczynski, J B; Meyer, B J

1998-11-12

Organisms in many phyla determine sexual fate by distinguishing one X chromosome from two. Here we use the model organism Caenorhabditis elegans to dissect such an X-chromosome-counting mechanism in molecular detail. In this nematode, several genes on the X chromosome called X signal elements communicate X-chromosome dose by controlling the activity of the sex-determination gene xol-1. xol-1 specifies male (XO) fate when active and hermaphrodite (XX) fate when inactive. The only X signal element described so far represses xol-1 post-transcriptionally, but xol-1 is repressed in XX animals by transcriptional and post-transcriptional mechanisms. Here we identify a nuclear-hormone-receptor homologue, SEX-1, that regulates the transcription of xol-1. We show that sex-1 is vital to X-chromosome counting: changing sex-1 gene dose in XX or XO embryos causes sexual transformation and death from inadequate dosage compensation (the hermaphrodite-specific process that equalizes X-gene expression between the sexes). The SEX-1 protein acts directly on xol-1, associating with its promoter in vivo and repressing xol-1 transcription in XX embryos. Thus, xol-1 is the direct molecular target of the primary sex-determination signal, and the dose of a nuclear hormone receptor helps to communicate X-chromosome number to determine nematode sex.

SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis.

PubMed

Muhar, Matthias; Ebert, Anja; Neumann, Tobias; Umkehrer, Christian; Jude, Julian; Wieshofer, Corinna; Rescheneder, Philipp; Lipp, Jesse J; Herzog, Veronika A; Reichholf, Brian; Cisneros, David A; Hoffmann, Thomas; Schlapansky, Moritz F; Bhat, Pooja; von Haeseler, Arndt; Köcher, Thomas; Obenauf, Anna C; Popow, Johannes; Ameres, Stefan L; Zuber, Johannes

2018-05-18

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.

PubMed

Takahashi, Melissa K; Watters, Kyle E; Gasper, Paul M; Abbott, Timothy R; Carlson, Paul D; Chen, Alan A; Lucks, Julius B

2016-06-01

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators. © 2016 Takahashi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A regulatory framework for shoot stem cell control integrating metabolic, transcriptional, and phytohormone signals.

PubMed

Schuster, Christoph; Gaillochet, Christophe; Medzihradszky, Anna; Busch, Wolfgang; Daum, Gabor; Krebs, Melanie; Kehle, Andreas; Lohmann, Jan U

2014-02-24

Plants continuously maintain pluripotent stem cells embedded in specialized tissues called meristems, which drive long-term growth and organogenesis. Stem cell fate in the shoot apical meristem (SAM) is controlled by the homeodomain transcription factor WUSCHEL (WUS) expressed in the niche adjacent to the stem cells. Here, we demonstrate that the bHLH transcription factor HECATE1 (HEC1) is a target of WUS and that it contributes to SAM function by promoting stem cell proliferation, while antagonizing niche cell activity. HEC1 represses the stem cell regulators WUS and CLAVATA3 (CLV3) and, like WUS, controls genes with functions in metabolism and hormone signaling. Among the targets shared by HEC1 and WUS are phytohormone response regulators, which we show to act as mobile signals in a universal feedback system. Thus, our work sheds light on the mechanisms guiding meristem function and suggests that the underlying regulatory system is far more complex than previously anticipated. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

PubMed Central

Zhao, Jian-Yuan; Liang, Lingli; Gu, Xiyao; Li, Zhisong; Wu, Shaogen; Sun, Linlin; Atianjoh, Fidelis E.; Feng, Jian; Mo, Kai; Jia, Shushan; Lutz, Brianna Marie; Bekker, Alex; Nestler, Eric J.; Tao, Yuan-Xiang

2017-01-01

Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG. PMID:28270689

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sung-Hak; Park, Jinah; Choi, Moon-Chang

DNA methyltransferases (DNMT) 3B is a de novo DNMT that represses transcription independent of DNMT activity. In order to gain a better insight into DNMT3B-mediated transcriptional repression, we performed a yeast two-hybrid analysis using DNMT3B as a bait. Of the various binding candidates, ZHX1, a member of zinc-finger and homeobox protein, was found to interact with DNMT3B in vivo and in vitro. N-terminal PWWP domain of DNMT3B was required for its interaction with homeobox motifs of ZHX1. ZHX1 contains nuclear localization signal at C-terminal homeobox motif, and both ZHX1 and DNMT3B were co-localized in nucleus. Furthermore, we found that ZHX1more » enhanced the transcriptional repression mediated by DNMT3B when DNMT3B is directly targeted to DNA. These results showed for First the direct linkage between DNMT and zinc-fingers homeoboxes protein, leading to enhanced gene silencing by DNMT3B.« less

ATRX Directs Binding of PRC2 to Xist RNA and Polycomb Targets

PubMed Central

Sarma, Kavitha; Cifuentes-Rojas, Catherine; Ergun, Ayla; del Rosario, Amanda; Jeon, Yesu; White, Forest; Sadreyev, Ruslan; Lee, Jeannie T.

2015-01-01

SUMMARY X chromosome inactivation (XCI) depends on the long noncoding RNA Xist and its recruitment of Polycomb Repressive Complex 2 (PRC2). PRC2 is also targeted to other sites throughout the genome to effect transcriptional repression. Using XCI as a model, we apply an unbiased proteomics approach to isolate Xist and PRC2 regulators and identified ATRX. ATRX unexpectedly functions as a high-affinity RNA-binding protein that directly interacts with RepA/Xist RNA to promote loading of PRC2 in vivo. Without ATRX, PRC2 cannot load onto Xist RNA nor spread in cis along the X chromosome. Moreover, epigenomic profiling reveals that genome-wide targeting of PRC2 depends on ATRX, as loss of ATRX leads to spatial redistribution of PRC2 and derepression of Polycomb responsive genes. Thus, ATRX is a required specificity determinant for PRC2 targeting and function. PMID:25417162

The Saccharomyces cerevisiae Cdk8 Mediator Represses AQY1 Transcription by Inhibiting Set1p-Dependent Histone Methylation.

PubMed

Law, Michael J; Finger, Michael A

2017-03-10

In the budding yeast Saccharomyces cerevisiae , nutrient depletion induces massive transcriptional reprogramming that relies upon communication between transcription factors, post-translational histone modifications, and the RNA polymerase II holoenzyme complex. Histone H3Lys4 methylation (H3Lys4 me), regulated by the Set1p-containing COMPASS methyltransferase complex and Jhd2p demethylase, is one of the most well-studied histone modifications. We previously demonstrated that the RNA polymerase II mediator components cyclin C-Cdk8p inhibit locus-specific H3Lys4 3me independently of Jhd2p Here, we identify loci subject to cyclin C- and Jhd2p-dependent histone H3Lys4 3me inhibition using chromatin immunoprecipitation (ChIP)-seq. We further characterized the independent and combined roles of cyclin C and Jhd2p in controlling H3Lys4 3me and transcription in response to fermentable and nonfermentable carbon at multiple loci. These experiments suggest that H3Lys4 3me alone is insufficient to induce transcription. Interestingly, we identified an unexpected role for cyclin C-Cdk8p in repressing AQY1 transcription, an aquaporin whose expression is normally induced during nutrient deprivation. These experiments, combined with previous work in other labs, support a two-step model in which cyclin C-Cdk8p mediate AQY1 transcriptional repression by stimulating transcription factor proteolysis and preventing Set1p recruitment to the AQY1 locus. Copyright © 2017 Law and Finger.

Thyroid Hormone Receptor β Suppression of RUNX2 is Mediated by Brahma Related Gene 1 Dependent Chromatin Remodeling.

PubMed

Gillis, Noelle E; Taber, Thomas H; Bolf, Eric L; Beaudet, Caitlin M; Tomczak, Jennifer A; White, Jeffrey H; Stein, Janet L; Stein, Gary S; Lian, Jane B; Frietze, Seth; Carr, Frances E

2018-05-09

Thyroid hormone receptor beta (TRβ) suppresses tumor growth through regulation of gene expression, yet the associated TRβ-mediated changes in chromatin assembly are not known. The chromatin ATPase Brahma Related Gene 1 (BRG1, SMARCA4), a key component of chromatin remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenesis and TRβ-mediated gene expression is unknown. We previously identified the oncogene runt-related transcription factor 2 (RUNX2) as a repressive target of TRβ. Here we report differential expression of BRG1 in non-malignant and malignant thyroid cells concordant with TRβ. BRG1 and TRβ have similar nuclear distribution patterns and significant co-localization. BRG1 interacts with TRβ and together are part of the regulatory complex at the RUNX2 promoter. Loss of BRG1 increases RUNX2 levels whereas re-introduction of TRβ and BRG1 synergistically decrease RUNX2 expression. RUNX2 promoter accessibility corresponded to RUNX2 expression levels. Inhibition of BRG1 activity ncreased accessibility of the RUNX2 promoter and corresponding expression. Our results reveal a novel mechanism of TRβ repression of oncogenic gene expression: TRβ recruitment of BRG1 to induce chromatin compaction and diminished RUNX2 expression. Therefore, BRG1-mediated chromatin remodeling may be obligatory for TRβ transcriptional repression and tumor suppressor function in thyroid tumorigenesis.

Mechanisms of iron regulation of luminescence in Vibrio fischeri.

PubMed Central

Haygood, M G; Nealson, K H

1985-01-01

Synthesis of luciferase (an autoinducible enzyme) is repressed by iron in the symbiotic bioluminescent bacterium Vibrio fischeri. Possible mechanisms of iron regulation of luciferase synthesis were tested with V. fischeri and with Escherichia coli clones containing plasmids carrying V. fischeri luminescence genes. Experiments were conducted in complete medium with and without the synthetic iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid). Comparison of the effect of ethylenediamine-di(o-hydroxyphenyl acetic acid) and another growth inhibitor, (2-n-heptyl-4-hydroxyquinoline-N-oxide), showed that iron repression is not due to inhibition of growth. A quantitative bioassay for autoinducer was developed with E. coli HB101 containing pJE411, a plasmid carrying V. fischeri luminescence genes with a transcriptional fusion between luxI and E. coli lacZ. Bioassay experiments showed no effect of iron on either autoinducer activity or production (before induction) or transcription of the lux operon. Ethylenediamine-di(o-hydroxyphenyl acetic acid) did not affect luciferase induction in E. coli strains with wild-type iron assimilation (ED8654) or impaired iron assimilation (RW193) bearing pJE202 (a plasmid with functional V. fischeri lux genes), suggesting that the genes responsible for the iron effect are missing or substituted in these clones. Two models are consistent with the data: (i) iron represses autoinducer transport, and (ii) iron acts through an autoinduction-independent regulatory system (e.g., an iron repressor). PMID:3920202

CsrA Represses Translation of sdiA, Which Encodes the N-Acylhomoserine-l-Lactone Receptor of Escherichia coli, by Binding Exclusively within the Coding Region of sdiA mRNA ▿ †

PubMed Central

Yakhnin, Helen; Baker, Carol S.; Berezin, Igor; Evangelista, Michael A.; Rassin, Alisa; Romeo, Tony; Babitzke, Paul

2011-01-01

The RNA binding protein CsrA is the central component of a conserved global regulatory system that activates or represses gene expression posttranscriptionally. In every known example of CsrA-mediated translational control, CsrA binds to the 5′ untranslated region of target transcripts, thereby repressing translation initiation and/or altering the stability of the RNA. Furthermore, with few exceptions, repression by CsrA involves binding directly to the Shine-Dalgarno sequence and blocking ribosome binding. sdiA encodes the quorum-sensing receptor for N-acyl-l-homoserine lactone in Escherichia coli. Because sdiA indirectly stimulates transcription of csrB, which encodes a small RNA (sRNA) antagonist of CsrA, we further explored the relationship between sdiA and the Csr system. Primer extension analysis revealed four putative transcription start sites within 85 nucleotides of the sdiA initiation codon. Potential σ70-dependent promoters were identified for each of these primer extension products. In addition, two CsrA binding sites were predicted in the initially translated region of sdiA. Expression of chromosomally integrated sdiA′-′lacZ translational fusions containing the entire promoter and CsrA binding site regions indicates that CsrA represses sdiA expression. The results from gel shift and footprint studies demonstrate that tight binding of CsrA requires both of these sites. Furthermore, the results from toeprint and in vitro translation experiments indicate that CsrA represses translation of sdiA by directly competing with 30S ribosomal subunit binding. Thus, this represents the first example of CsrA preventing translation by interacting solely within the coding region of an mRNA target. PMID:21908661

Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

PubMed

Pragman, Alexa A; Yarwood, Jeremy M; Tripp, Timothy J; Schlievert, Patrick M

2004-04-01

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

Sulfoxidation Regulation of Musa acuminata Calmodulin (MaCaM) Influences the Functions of MaCaM-Binding Proteins.

PubMed

Jiang, Guoxiang; Wu, Fuwang; Li, Zhiwei; Li, Taotao; Gupta, Vijai Kumar; Duan, Xuewu; Jiang, Yueming

2018-06-01

Sulfoxidation of methionine in proteins by reactive oxygen species can cause conformational alteration or functional impairment, and can be reversed by methionine sulfoxide reductase (Msr). Currently, only a few potential Msr substrates have been confirmed in higher plants. Here, we investigated Msr-mediated sulfoxidation regulation of calmodulin (CaM) and its underlying biological significance in relation to banana fruit ripening and senescence. Expression of MaCaM1 and MaMsrA7 was up-regulated with increased ripening and senescence. We verified that MaCaM1 interacts with MaMsrA7 in vitro and in vivo, and sulfoxidated MaCaM1 could be partly repaired by MaMsrA7 (MaMsrA7 reduces oxidized residues Met77 and Met110 in MaCaM1). Furthermore, we investigated two known CaM-binding proteins, catalase (MaCAT1) and MaHY5-1. MaHY5-1 acts as a transcriptional repressor of carotenoid biosynthesis-related genes (MaPSY1, MaPSY2 and MaPSY3) in banana fruit. MaCaM1 could enhance the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1 toward MaPSY2. Mimicked sulfoxidation in MaCaM1 did not affect the physical interactions of the protein with MaHY5-1 and MaCAT1, but reduced the catalytic activity of MaCAT1 and the transcriptional repression activity of MaHY5-1. Our data suggest that sulfoxidation modification in MaCaM1 by MaMsrA7 regulates antioxidant response and gene transcription, thereby being involved in regulation of ripening and senescence of banana fruit.

A role for Candida albicans superoxide dismutase enzymes in glucose signaling.

PubMed

Broxton, Chynna N; He, Bixi; Bruno, Vincent M; Culotta, Valeria C

2018-01-01

The Saccharomyces cerevisiae and Candida albicans yeasts have evolved to differentially use glucose for fermentation versus respiration. S. cerevisiae is Crabtree positive, where glucose represses respiration and promotes fermentation, while the opportunistic fungal pathogen C. albicans is Crabtree negative and does not repress respiration with glucose. We have previously shown that glucose control in S. cerevisiae involves the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1), where H 2 O 2 generated by SOD1 stabilizes the casein kinase YCK1 for glucose sensing. We now demonstrate that C. albicans SODs also participate in glucose regulation. C. albicans expresses two cytosolic SODs, Cu/Zn SOD1 and Mn containing SOD3, and both complemented a S. cerevisiae sod1Δ mutant in stabilizing YCK1. Moreover, in C. albicans cells, both SODs functioned to repress glucose transporter genes in response to glucose. However, the action of SODs in glucose control has diverged in the two yeasts. In S. cerevisiae, SOD1 specifically functions in the glucose sensing pathway involving YCK1 and the RGT1 repressor, but the analogous YCK/RGT1 pathway in C. albicans shows no control by SOD enzymes. Instead C. albicans SODs work in the glucose repression pathway involving the MIG1 transcriptional repressor. In C. albicans, the SODs repress glucose uptake, while in S. cerevisiae, SOD1 activates glucose uptake, in accordance with the divergent modes for glucose utilization in these two distantly related yeasts. Copyright © 2017 Elsevier Inc. All rights reserved.

Cardiac Expression of ms1/STARS, a Novel Gene Involved in Cardiac Development and Disease, Is Regulated by GATA4

PubMed Central

Kobayashi, Satoru; Peterson, Richard E.; He, Aibin; Motterle, Anna; Samani, Nilesh J.; Menick, Donald R.; Pu, William T.; Liang, Qiangrong

2012-01-01

Ms1/STARS is a novel muscle-specific actin-binding protein that specifically modulates the myocardin-related transcription factor (MRTF)-serum response factor (SRF) regulatory axis within striated muscle. This ms1/STARS-dependent regulatory axis is of central importance within the cardiac gene regulatory network and has been implicated in cardiac development and postnatal cardiac function/homeostasis. The dysregulation of ms1/STARS is associated with and causative of pathological cardiac phenotypes, including cardiac hypertrophy and cardiomyopathy. In order to gain an understanding of the mechanisms governing ms1/STARS expression in the heart, we have coupled a comparative genomic in silico analysis with reporter, gain-of-function, and loss-of-function approaches. Through this integrated analysis, we have identified three evolutionarily conserved regions (ECRs), α, SINA, and DINA, that act as cis-regulatory modules and confer differential cardiac cell-specific activity. Two of these ECRs, α and DINA, displayed distinct regulatory sensitivity to the core cardiac transcription factor GATA4. Overall, our results demonstrate that within embryonic, neonatal, and adult hearts, GATA4 represses ms1/STARS expression with the pathologically associated depletion of GATA4 (type 1/type 2 diabetic models), resulting in ms1/STARS upregulation. This GATA4-dependent repression of ms1/STARS expression has major implications for MRTF-SRF signaling in the context of cardiac development and disease. PMID:22431517

VEGF and VEGFB Play Balancing Roles in Adipose Differentiation, Gene Expression, and Function.

PubMed

Jin, Honghong; Li, Dan; Wang, Xutong; Jia, Jia; Chen, Yang; Yao, Yapeng; Zhao, Chunlan; Lu, Xiaodan; Zhang, Shujie; Togo, Jacques; Ji, Yan; Zhang, Luqing; Feng, Xuechao; Zheng, Yaowu

2018-05-01

Obesity is the result of abnormal adipose development and energy metabolism. Using vascular endothelial growth factor (VEGF) B-knockout and inducible VEGF downregulation mouse models, we have shown that VEGFB inactivation caused expansion of white adipose, whitening of brown adipose, an increase in fat accumulation, and a reduction in energy consumption. At the same time, expression of the white adipose-associated genes was increased and brown adipose-associated genes decreased. VEGF repression, in contrast, induced brown adipose expansion and brown adipocyte development in white adipose, increased energy expenditure, upregulated brown adipose-associated genes, and downregulated white adipose-associated genes. When VEGFB-knockout and VEGF-repressed mice are crossed together, VEGF and VEGFB can counteractively regulate large numbers of genes and efficiently reverse each other's roles. These genes, under counteractive VEGF and VEGFB regulations, include transcription factors, adhesion molecules, and metabolic enzymes. This balancing role is confirmed by morphologic and functional changes. This study reports that VEGF and VEGFB counteractively regulate adipose development and function in energy metabolism.

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.

Genome wide interactions of wild-type and activator bypass forms of σ54.

PubMed

Schaefer, Jorrit; Engl, Christoph; Zhang, Nan; Lawton, Edward; Buck, Martin

2015-09-03

Enhancer-dependent transcription involving the promoter specificity factor σ(54) is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, σ(54)-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ(54) promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ(54) were evident, indicating loss of rpoN (encoding σ(54)) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ(54)-dependence in vivo not readily correlated with conventional σ(54) binding-sites. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome wide interactions of wild-type and activator bypass forms of σ54

PubMed Central

Schaefer, Jorrit; Engl, Christoph; Zhang, Nan; Lawton, Edward; Buck, Martin

2015-01-01

Enhancer-dependent transcription involving the promoter specificity factor σ54 is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, σ54-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ54 promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ54 were evident, indicating loss of rpoN (encoding σ54) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ54-dependence in vivo not readily correlated with conventional σ54 binding-sites. PMID:26082500

Redirection of the Respiro-Fermentative Flux Distribution in Saccharomyces cerevisiae by Overexpression of the Transcription Factor Hap4p

PubMed Central

Blom, Jolanda; De Mattos, M. Joost Teixeira; Grivell, Leslie A.

2000-01-01

Reduction of aerobic fermentation on sugars by altering the fermentative/oxidative balance is of significant interest for optimization of industrial production of Saccharomyces cerevisiae. Glucose control of oxidative metabolism in baker's yeast is partly mediated through transcriptional regulation of the Hap4p subunit of the Hap2/3/4/5p transcriptional activator complex. To alleviate glucose repression of oxidative metabolism, we constructed a yeast strain with constitutively elevated levels of Hap4p. Genetic analysis of expression levels of glucose-repressed genes and analysis of respiratory capacity showed that Hap4p overexpression (partly) relieves glucose repression of respiration. Analysis of the physiological properties of the Hap4p overproducer in batch cultures in fermentors (aerobic, glucose excess) has shown that the metabolism of this strain is more oxidative than in the wild-type strain, resulting in a significant reduced ethanol production and improvement of growth rate and a 40% gain in biomass yield. Our results show that modification of one or more transcriptional regulators can be a powerful and a widely applicable tool for redirection of metabolic fluxes in microorganisms. PMID:10788368

The transcriptional regulator, CosR, controls compatible solute biosynthesis and transport, motility and biofilm formation in Vibrio cholerae.

PubMed

Shikuma, Nicholas J; Davis, Kimberly R; Fong, Jiunn N C; Yildiz, Fitnat H

2013-05-01

Vibrio cholerae inhabits aquatic environments and colonizes the human digestive tract to cause the disease cholera. In these environments, V. cholerae copes with fluctuations in salinity and osmolarity by producing and transporting small, organic, highly soluble molecules called compatible solutes, which counteract extracellular osmotic pressure. Currently, it is unclear how V. cholerae regulates the expression of genes important for the biosynthesis or transport of compatible solutes in response to changing salinity or osmolarity conditions. Through a genome-wide transcriptional analysis of the salinity response of V. cholerae, we identified a transcriptional regulator we name CosR for compatible solute regulator. The expression of cosR is regulated by ionic strength and not osmolarity. A transcriptome analysis of a ΔcosR mutant revealed that CosR represses genes involved in ectoine biosynthesis and compatible solute transport in a salinity-dependent manner. When grown in salinities similar to estuarine environments, CosR activates biofilm formation and represses motility independently of its function as an ectoine regulator. This is the first study to characterize a compatible solute regulator in V. cholerae and couples the regulation of osmotic tolerance with biofilm formation and motility. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

The Emerging Role of Epigenetics in the Regulation of Female Puberty.

PubMed

Lomniczi, Alejandro; Ojeda, Sergio R

2016-01-01

In recent years the pace of discovering the molecular and genetic underpinnings of the pubertal process has accelerated considerably. Genes required for human puberty to occur have been identified and evidence has been provided suggesting that the initiation of puberty requires coordinated changes in the output of a multiplicity of genes organized into functional networks. Recent evidence suggests that a dual mechanism of epigenetic regulation affecting the transcriptional activity of neurons involved in stimulating gonadotropin-releasing hormone release plays a fundamental role in the timing of puberty. The Polycomb group (PcG) of transcriptional silencers appears to be a major component of the repressive arm of this mechanism. PcG proteins prevent the premature initiation of female puberty by silencing the Kiss1 gene in kisspeptin neurons of the arcuate nucleus (ARC) of the hypothalamus. Because the abundance of histone marks either catalyzed by--or associated with--the Trithorax group (TrxG) of transcriptional activators increases at the time when PcG control subsides, it appears that the TrxG complex is the counteracting partner of PcG-mediated gene silencing. In this chapter, we discuss the concept that a switch from epigenetic repression to activation within ARC kisspeptin neurons is a core mechanism underlying the initiation of female puberty. © 2016 S. Karger AG, Basel.

The Emerging Role of Epigenetics in the Regulation of Female Puberty

PubMed Central

Lomniczi, Alejandro; Ojeda, Sergio R.

2016-01-01

In recent years the pace of discovering the molecular and genetic underpinnings of the pubertal process has accelerated considerably. Genes required for human puberty to occur have been identified and evidence has been provided suggesting that the initiation of puberty requires coordinated changes in the output of a multiplicity of genes organized into functional networks. Recent evidence suggests that a dual mechanism of epigenetic regulation affecting the transcriptional activity of neurons involved in stimulating gonadotropin-releasing hormone release plays a fundamental role in the timing of puberty. The Polycomb group (PcG) of transcriptional silencers appears to be a major component of the repressive arm of this mechanism. PcG proteins prevent the premature initiation of female puberty by silencing the Kiss1 gene in kisspeptin neurons of the arcuate nucleus (ARC) of the hypothalamus. Because the abundance of histone marks either catalyzed by – or associated with – the Trithorax group (TrxG) of transcriptional activators increases at the time when PcG control subsides, it appears that the TrxG complex is the counteracting partner of PcG-mediated gene silencing. In this chapter, we discuss the concept that a switch from epigenetic repression to activation within ARC kisspeptin neurons is a core mechanism underlying the initiation of female puberty. PMID:26680569

Mediator complex cooperatively regulates transcription of retinoic acid target genes with Polycomb Repressive Complex 2 during neuronal differentiation.

PubMed

Fukasawa, Rikiya; Iida, Satoshi; Tsutsui, Taiki; Hirose, Yutaka; Ohkuma, Yoshiaki

2015-11-01

The Mediator complex (Mediator) plays key roles in transcription and functions as the nexus for integration of various transcriptional signals. Previously, we screened for Mediator cyclin-dependent kinase (CDK)-interacting factors and identified three proteins related to chromatin regulation. One of them, SUZ12 is required for both stability and activity of Polycomb Repressive Complex 2 (PRC2). PRC2 primarily suppresses gene expression through histone H3 lysine 27 trimethylation, resulting in stem cell maintenance and differentiation; perturbation of this process leads to oncogenesis. Recent work showed that Mediator contributes to the embryonic stem cell state through DNA loop formation, which is strongly associated with chromatin architecture; however, it remains unclear how Mediator regulates gene expression in cooperation with chromatin regulators (i.e. writers, readers and remodelers). We found that Mediator CDKs interact directly with the PRC2 subunit EZH2, as well as SUZ12. Known PRC2 target genes were deregulated by Mediator CDK knockdown during neuronal differentiation, and both Mediator and PRC2 complexes co-occupied the promoters of developmental genes regulated by retinoic acid. Our results provide a mechanistic link between Mediator and PRC2 during neuronal differentiation. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Tristetraprolin Represses Estrogen Receptor α Transactivation in Breast Cancer Cells*

PubMed Central

Barrios-García, Tonatiuh; Tecalco-Cruz, Angeles; Gómez-Romero, Vania; Reyes-Carmona, Sandra; Meneses-Morales, Iván; León-Del-Río, Alfonso

2014-01-01

Estrogen receptor α (ERα) mediates the effects of 17β-estradiol (E2) in normal mammary gland, and it is a key participant in breast cancer tumor development. ERα transactivation activity is mediated by the synergistic interaction of two domains designated AF1 and AF2. The function of AF2 is to recruit coactivator and corepressor proteins that allow ERα to oscillate between the roles of transcriptional activator and repressor. In contrast, the mechanism responsible for AF-1 transcriptional activity is not completely understood. In this study, we identified tristetraproline (TTP) as a novel ERα-associated protein. TTP expression in MCF7 cells repressed ERα transactivation and reduced MCF7 cell proliferation and the ability of the cells to form tumors in a mouse model. We show that TTP transcriptional activity is mediated through its recruitment to the promoter region of ERα target genes and its interaction with histone deacetylases, in particular with HDAC1. TTP expression attenuates the coactivating activity of SRC-1, suggesting that exchange between TTP and other coactivators may play an important role in fine-tuning ERα transactivation. These results indicate that TTP acts as a bona fide ERα corepressor and suggest that this protein may be a contributing factor in the development of E2-dependent tumors in breast cancer. PMID:24737323

Orphan nuclear receptor TLX recruits histone deacetylases to repress transcription and regulate neural stem cell proliferation

PubMed Central

Sun, GuoQiang; Yu, Ruth T.; Evans, Ronald M.; Shi, Yanhong

2007-01-01

TLX is a transcription factor that is essential for neural stem cell proliferation and self-renewal. However, the molecular mechanism of TLX-mediated neural stem cell proliferation and self-renewal is largely unknown. We show here that TLX recruits histone deacetylases (HDACs) to its downstream target genes to repress their transcription, which in turn regulates neural stem cell proliferation. TLX interacts with HDAC3 and HDAC5 in neural stem cells. The HDAC5-interaction domain was mapped to TLX residues 359–385, which contains a conserved nuclear receptor–coregulator interaction motif IXXLL. Both HDAC3 and HDAC5 have been shown to be recruited to the promoters of TLX target genes along with TLX in neural stem cells. Recruitment of HDACs led to transcriptional repression of TLX target genes, the cyclin-dependent kinase inhibitor, p21CIP1/WAF1(p21), and the tumor suppressor gene, pten. Either inhibition of HDAC activity or knockdown of HDAC expression led to marked induction of p21 and pten gene expression and dramatically reduced neural stem cell proliferation, suggesting that the TLX-interacting HDACs play an important role in neural stem cell proliferation. Moreover, expression of a TLX peptide containing the minimal HDAC5 interaction domain disrupted the TLX–HDAC5 interaction. Disruption of this interaction led to significant induction of p21 and pten gene expression and to dramatic inhibition of neural stem cell proliferation. Taken together, these findings demonstrate a mechanism for neural stem cell proliferation through transcriptional repression of p21 and pten gene expression by TLX–HDAC interactions. PMID:17873065

A feedback regulatory model for RifQ-mediated repression of rifamycin export in Amycolatopsis mediterranei.

PubMed

Lei, Chao; Wang, Jingzhi; Liu, Yuanyuan; Liu, Xinqiang; Zhao, Guoping; Wang, Jin

2018-01-29

Due to the important role of rifamycin in curing tuberculosis infection, the study on rifamycin has never been stopped. Although RifZ, which locates within the rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for rifamycin biosynthesis, little is known about the regulation of rifamycin export. In this work, we proved that the expression of the rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP's transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP's transcription in vivo. Moreover, rifamycin B (the end product of rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of rifamycin export, reducing the intracellular concentration of rifamycin B as well as its toxicity against the host. Here, we proved that the export of rifamycin B was repressed by RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could be specifically relieved by rifamycin B, the end product of rifamycin biosynthesis, based on which a feedback model was proposed for regulation of rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter.

On the presence and role of human gene-body DNA methylation

PubMed Central

Jjingo, Daudi; Conley, Andrew B.; Yi, Soojin V.; Lunyak, Victoria V.; Jordan, I. King

2012-01-01

DNA methylation of promoter sequences is a repressive epigenetic mark that down-regulates gene expression. However, DNA methylation is more prevalent within gene-bodies than seen for promoters, and gene-body methylation has been observed to be positively correlated with gene expression levels. This paradox remains unexplained, and accordingly the role of DNA methylation in gene-bodies is poorly understood. We addressed the presence and role of human gene-body DNA methylation using a meta-analysis of human genome-wide methylation, expression and chromatin data sets. Methylation is associated with transcribed regions as genic sequences have higher levels of methylation than intergenic or promoter sequences. We also find that the relationship between gene-body DNA methylation and expression levels is non-monotonic and bell-shaped. Mid-level expressed genes have the highest levels of gene-body methylation, whereas the most lowly and highly expressed sets of genes both have low levels of methylation. While gene-body methylation can be seen to efficiently repress the initiation of intragenic transcription, the vast majority of methylated sites within genes are not associated with intragenic promoters. In fact, highly expressed genes initiate the most intragenic transcription, which is inconsistent with the previously held notion that gene-body methylation serves to repress spurious intragenic transcription to allow for efficient transcriptional elongation. These observations lead us to propose a model to explain the presence of human gene-body methylation. This model holds that the repression of intragenic transcription by gene-body methylation is largely epiphenomenal, and suggests that gene-body methylation levels are predominantly shaped via the accessibility of the DNA to methylating enzyme complexes. PMID:22577155

I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions.

PubMed

Kusano, Shuichi; Yoshimitsu, Makoto; Hachiman, Miho; Ikeda, Masanori

2015-12-01

The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. Copyright © 2015 Elsevier Inc. All rights reserved.

Primetime for Learning Genes.

PubMed

Keifer, Joyce

2017-02-11

Learning genes in mature neurons are uniquely suited to respond rapidly to specific environmental stimuli. Expression of individual learning genes, therefore, requires regulatory mechanisms that have the flexibility to respond with transcriptional activation or repression to select appropriate physiological and behavioral responses. Among the mechanisms that equip genes to respond adaptively are bivalent domains. These are specific histone modifications localized to gene promoters that are characteristic of both gene activation and repression, and have been studied primarily for developmental genes in embryonic stem cells. In this review, studies of the epigenetic regulation of learning genes in neurons, particularly the brain-derived neurotrophic factor gene ( BDNF ), by methylation/demethylation and chromatin modifications in the context of learning and memory will be highlighted. Because of the unique function of learning genes in the mature brain, it is proposed that bivalent domains are a characteristic feature of the chromatin landscape surrounding their promoters. This allows them to be "poised" for rapid response to activate or repress gene expression depending on environmental stimuli.

H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus.

PubMed

Wang, Yan; Zhang, Yiquan; Yin, Zhe; Wang, Jie; Zhu, Yongzhe; Peng, Haoran; Zhou, Dongsheng; Qi, Zhongtian; Yang, Wenhui

2018-01-01

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ 28 -dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.

A functional genomics strategy reveals Rora as a component of the mammalian circadian clock.

PubMed

Sato, Trey K; Panda, Satchidananda; Miraglia, Loren J; Reyes, Teresa M; Rudic, Radu D; McNamara, Peter; Naik, Kinnery A; FitzGerald, Garret A; Kay, Steve A; Hogenesch, John B

2004-08-19

The mammalian circadian clock plays an integral role in timing rhythmic physiology and behavior, such as locomotor activity, with anticipated daily environmental changes. The master oscillator resides within the suprachiasmatic nucleus (SCN), which can maintain circadian rhythms in the absence of synchronizing light input. Here, we describe a genomics-based approach to identify circadian activators of Bmal1, itself a key transcriptional activator that is necessary for core oscillator function. Using cell-based functional assays, as well as behavioral and molecular analyses, we identified Rora as an activator of Bmal1 transcription within the SCN. Rora is required for normal Bmal1 expression and consolidation of daily locomotor activity and is regulated by the core clock in the SCN. These results suggest that opposing activities of the orphan nuclear receptors Rora and Rev-erb alpha, which represses Bmal1 expression, are important in the maintenance of circadian clock function.

Nitrate Controls Root Development through Posttranscriptional Regulation of the NRT1.1/NPF6.3 Transporter/Sensor1

PubMed Central

Perrine-Walker, Francine; Rochette, Juliette; Martinière, Alexandre; Bach, Lien; Gojon, Alain

2016-01-01

Plants are able to modulate root growth and development to optimize their nitrogen nutrition. In Arabidopsis (Arabidopsis thaliana), the adaptive root response to nitrate (NO3−) depends on the NRT1.1/NPF6.3 transporter/sensor. NRT1.1 represses emergence of lateral root primordia (LRPs) at low concentration or absence of NO3− through its auxin transport activity that lowers auxin accumulation in LR. However, these functional data strongly contrast with the known transcriptional regulation of NRT1.1, which is markedly repressed in LRPs in the absence of NO3−. To explain this discrepancy, we investigated in detail the spatiotemporal expression pattern of the NRT1.1 protein during LRP development and combined local transcript analysis with the use of transgenic lines expressing tagged NRT1.1 proteins. Our results show that although NO3− stimulates NRT1.1 transcription and probably mRNA stability both in primary root tissues and in LRPs, it acts differentially on protein accumulation, depending on the tissues considered with stimulation in cortex and epidermis of the primary root and a strong repression in LRPs and to a lower extent at the primary root tip. This demonstrates that NRT1.1 is strongly regulated at the posttranscriptional level by tissue-specific mechanisms. These mechanisms are crucial for controlling the large palette of adaptive responses to NO3− mediated by NRT1.1 as they ensure that the protein is present in the proper tissue under the specific conditions where it plays a signaling role in this particular tissue. PMID:27543115

Spontaneous mutations in CYC8 and MIG1 suppress the short chronological lifespan of budding yeast lacking SNF1/AMPK

PubMed Central

Maqani, Nazif; Fine, Ryan D.; Shahid, Mehreen; Li, Mingguang; Enriquez-Hesles, Elisa; Smith, Jeffrey S.

2018-01-01

Chronologically aging yeast cells are prone to adaptive regrowth, whereby mutants with a survival advantage spontaneously appear and re-enter the cell cycle in stationary phase cultures. Adaptive regrowth is especially noticeable with short-lived strains, including those defective for SNF1, the homolog of mammalian AMP-activated protein kinase (AMPK). SNF1 becomes active in response to multiple environmental stresses that occur in chronologically aging cells, including glucose depletion and oxidative stress. SNF1 is also required for the extension of chronological lifespan (CLS) by caloric restriction (CR) as defined as limiting glucose at the time of culture inoculation. To identify specific downstream SNF1 targets responsible for CLS extension during CR, we screened for adaptive regrowth mutants that restore chronological longevity to a short-lived snf1∆ parental strain. Whole genome sequencing of the adapted mutants revealed missense mutations in TPR motifs 9 and 10 of the transcriptional co-repressor Cyc8 that specifically mediate repression through the transcriptional repressor Mig1. Another mutation occurred in MIG1 itself, thus implicating the activation of Mig1-repressed genes as a key function of SNF1 in maintaining CLS. Consistent with this conclusion, the cyc8 TPR mutations partially restored growth on alternative carbon sources and significantly extended CLS compared to the snf1∆ parent. Furthermore, cyc8 TPR mutations reactivated multiple Mig1-repressed genes, including the transcription factor gene CAT8, which is responsible for activating genes of the glyoxylate and gluconeogenesis pathways. Deleting CAT8 completely blocked CLS extension by the cyc8 TPR mutations on CLS, identifying these pathways as key Snf1-regulated CLS determinants.

Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

PubMed

Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

2013-12-06

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation*

PubMed Central

Almenar-Queralt, Angels; Kim, Sonia N.; Benner, Christopher; Herrera, Cheryl M.; Kang, David E.; Garcia-Bassets, Ivan; Goldstein, Lawrence S. B.

2013-01-01

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment. PMID:24145027

HSI2/VAL1 Silences AGL15 to Regulate the Developmental Transition from Seed Maturation to Vegetative Growth in Arabidopsis[OPEN

PubMed Central

Abdelmageed, Haggag; Kang, Miyoung

2018-01-01

Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus. PMID:29475938

The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates.

PubMed

Gamble, Matthew J; Erdjument-Bromage, Hediye; Tempst, Paul; Freedman, Leonard P; Fisher, Robert P

2005-01-01

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

The transcriptional PPARβ/δ network in human macrophages defines a unique agonist-induced activation state

PubMed Central

Adhikary, Till; Wortmann, Annika; Schumann, Tim; Finkernagel, Florian; Lieber, Sonja; Roth, Katrin; Toth, Philipp M.; Diederich, Wibke E.; Nist, Andrea; Stiewe, Thorsten; Kleinesudeik, Lara; Reinartz, Silke; Müller-Brüsselbach, Sabine; Müller, Rolf

2015-01-01

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor with established metabolic functions, whereas its anti-inflammatory function is poorly understood. To address this issue, we determined the global PPARβ/δ-regulated signaling network in human monocyte-derived macrophages. Besides cell type-independent, canonical target genes with metabolic and immune regulatory functions we identified a large number of inflammation-associated NFκB and STAT1 target genes that are repressed by agonists. Accordingly, PPARβ/δ agonists inhibited the expression of multiple pro-inflammatory mediators and induced an anti-inflammatory, IL-4-like morphological phenotype. Surprisingly, bioinformatic analyses also identified immune stimulatory effects. Consistent with this prediction, PPARβ/δ agonists enhanced macrophage survival under hypoxic stress and stimulated CD8+ T cell activation, concomitantly with the repression of immune suppressive target genes and their encoded products CD274 (PD-1 ligand), CD32B (inhibitory Fcγ receptor IIB) and indoleamine 2,3-dioxygenase 1 (IDO-1), as well as a diminished release of the immune suppressive IDO-1 metabolite kynurenine. Comparison with published data revealed a significant overlap of the PPARβ/δ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines. Our findings indicate that PPARβ/δ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARβ/δ in immune regulation. PMID:25934804

HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes.

PubMed

Morero, Natalia R; Botti, Horacio; Nitta, Kazuhiro R; Carrión, Federico; Obal, Gonzalo; Picardeau, Mathieu; Buschiazzo, Alejandro

2014-10-01

Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways, some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO - haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mechanistic model for its dual regulatory role. © 2014 John Wiley & Sons Ltd.

ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons.

PubMed

Noh, Kyung-Min; Maze, Ian; Zhao, Dan; Xiang, Bin; Wenderski, Wendy; Lewis, Peter W; Shen, Li; Li, Haitao; Allis, C David

2015-06-02

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of "repressive" histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX's ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this "methyl/phos" switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

Exploration of G-quadruplex function in c-Myb gene and its transcriptional regulation by topotecan.

PubMed

Li, Fangyuan; Zhou, Jiang; Xu, Ming; Yuan, Gu

2018-02-01

Our bioinformatics research shows that there are four G-rich sequences (S1-S4) in the upstream region of the transcription start site of c-Myb gene, and we have proved that these sequences have the ability to form G-quadruplex structures. This work mainly focuses on G-quadruplex function, recognition and transcription regulation in c-Myb gene, revealing a novel regulatory element in c-Myb proximal promoter region, and its transcription regulation by G-quadruplex binder. The research has identified that the enhancer effect in c-Myb transcription was primarily affected by the G-quadruplex formed by S1 sequence, and the up-regulation effect may due to the removal of repressive progress of MZF-1 by stabilizing G-quadruplex. Attentions were being paid to the development of G-quadruplex binders for selective recognition, and topotecan was found to have high binding affinity in vitro and could effectively affect the c-Myb transcription activities in cells. The regulation of G-quadruplex with binders in transcriptional, translational levels by Q-RT-PCR and western blot was in expectation of providing a strategy for gene expression modulation. In conclusion, our study revealed a G-quadruplex structure in c-Myb proximal promoter region, which was of great importance in the regulation of c-Myb function. Copyright © 2017 Elsevier B.V. All rights reserved.

Cooperative gene regulation by microRNA pairs and their identification using a computational workflow

PubMed Central

Schmitz, Ulf; Lai, Xin; Winter, Felix; Wolkenhauer, Olaf; Vera, Julio; Gupta, Shailendra K.

2014-01-01

MicroRNAs (miRNAs) are an integral part of gene regulation at the post-transcriptional level. Recently, it has been shown that pairs of miRNAs can repress the translation of a target mRNA in a cooperative manner, which leads to an enhanced effectiveness and specificity in target repression. However, it remains unclear which miRNA pairs can synergize and which genes are target of cooperative miRNA regulation. In this paper, we present a computational workflow for the prediction and analysis of cooperating miRNAs and their mutual target genes, which we refer to as RNA triplexes. The workflow integrates methods of miRNA target prediction; triplex structure analysis; molecular dynamics simulations and mathematical modeling for a reliable prediction of functional RNA triplexes and target repression efficiency. In a case study we analyzed the human genome and identified several thousand targets of cooperative gene regulation. Our results suggest that miRNA cooperativity is a frequent mechanism for an enhanced target repression by pairs of miRNAs facilitating distinctive and fine-tuned target gene expression patterns. Human RNA triplexes predicted and characterized in this study are organized in a web resource at www.sbi.uni-rostock.de/triplexrna/. PMID:24875477

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila

PubMed Central

Xiong, Xiao-Peng; Chang, Kung-Yen; Ren, Xingjie; Ni, Jian-Quan; Rana, Tariq M.; Zhou, Rui

2016-01-01

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity. PMID:27893816

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.

PubMed

Xiong, Xiao-Peng; Kurthkoti, Krishna; Chang, Kung-Yen; Li, Jian-Liang; Ren, Xingjie; Ni, Jian-Quan; Rana, Tariq M; Zhou, Rui

2016-11-01

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.

Repression of chimeric transcripts emanating from endogenous retrotransposons by a sequence-specific transcription factor

PubMed Central

2014-01-01

Background Retroviral elements are pervasively transcribed and dynamically regulated during development. While multiple histone- and DNA-modifying enzymes have broadly been associated with their global silencing, little is known about how the many diverse retroviral families are each selectively recognized. Results Here we show that the zinc finger protein Krüppel-like Factor 3 (KLF3) specifically silences transcription from the ORR1A0 long terminal repeat in murine fetal and adult erythroid cells. In the absence of KLF3, we detect widespread transcription from ORR1A0 elements driven by the master erythroid regulator KLF1. In several instances these aberrant transcripts are spliced to downstream genic exons. One such chimeric transcript produces a novel, dominant negative isoform of PU.1 that can induce erythroid differentiation. Conclusions We propose that KLF3 ensures the integrity of the murine erythroid transcriptome through the selective repression of a particular retroelement and is likely one of multiple sequence-specific factors that cooperate to achieve global silencing. PMID:24946810

Oncogenic deregulation of NKL homeobox gene MSX1 in mantle cell lymphoma.

PubMed

Nagel, Stefan; Ehrentraut, Stefan; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

2014-08-01

NKL homeobox gene MSX1 is physiologically expressed during embryonic hematopoiesis. Here, we detected MSX1 overexpression in three examples of mantle cell lymphoma (MCL) and one of acute myeloid leukemia (AML) by screening 96 leukemia/lymphoma cell lines via microarray profiling. Moreover, in silico analysis identified significant overexpression of MSX1 in 3% each of patients with MCL and AML, confirming aberrant activity in subsets of both types of malignancies. Comparative expression profiling analysis and subsequent functional studies demonstrated overexpression of histone acetyltransferase PHF16 together with transcription factors FOXC1 and HLXB9 as activators of MSX1 transcription. Additionally, we identified regulation of cyclin D1/CCND1 by MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization in MCL cells showed that t(11;14)(q13;q32) results in detachment of CCND1 from its corresponding repressive MSX1 binding site. Taken together, we uncovered regulators and targets of homeobox gene MSX1 in leukemia/lymphoma cells, supporting the view of a recurrent genetic network that is reactivated in malignant transformation.

Histone Deacetylase Inhibitors Repress Tumoral Expression of the Proinvasive Factor RUNX2.

PubMed

Sancisi, Valentina; Gandolfi, Greta; Ambrosetti, Davide Carlo; Ciarrocchi, Alessia

2015-05-01

Aberrant reactivation of embryonic pathways occurs commonly in cancer. The transcription factor RUNX2 plays a fundamental role during embryogenesis and is aberrantly reactivated during progression and metastasization of different types of human tumors. In this study, we attempted to dissect the molecular mechanisms governing RUNX2 expression and its aberrant reactivation. We identified a new regulatory enhancer element, located within the RUNX2 gene, which is responsible for the activation of the RUNX2 promoter and for the regulation of its expression in cancer cells. Furthermore, we have shown that treatment with the anticancer compounds histone deacetylase inhibitor (HDACi) results in a profound inhibition of RUNX2 expression, which is determined by the disruption of the transcription-activating complex on the identified enhancer. These data envisage a possible targeting strategy to counteract the oncongenic function of RUNX2 in cancer cells and provide evidence that the cytotoxic activity of HDACi in cancer is not only dependent on the reactivation of silenced oncosuppressors but also on the repression of oncogenic factors that are necessary for survival and progression. ©2015 American Association for Cancer Research.

Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function.

PubMed

Sakamaki, Jun-Ichi; Wilkinson, Simon; Hahn, Marcel; Tasdemir, Nilgun; O'Prey, Jim; Clark, William; Hedley, Ann; Nixon, Colin; Long, Jaclyn S; New, Maria; Van Acker, Tim; Tooze, Sharon A; Lowe, Scott W; Dikic, Ivan; Ryan, Kevin M

2017-05-18

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING

PubMed Central

Theodosiou, Maria; Monaghan, James R; Spencer, Michael L; Voss, S Randal; Noonan, Daniel J

2009-01-01

Retinoic acid, a key morphogen in early vertebrate development and tissue regeneration, mediates its effects through the binding of receptors that act as ligand-induced transcription factors. These binding events function to recruit an array of transcription co-regulatory proteins to specific gene promoters. One such co-regulatory protein, neuronal proliferation and differentiation control-1 (NPDC-1), is broadly expressed during mammalian development and functions as an in vitro repressor of retinoic acid receptor (RAR)-mediated transcription. To obtain comparative and developmental insights about NPDC-1 function, we cloned the axolotl (Ambystoma mexicanum) orthologue and measured transcript abundances among tissues sampled during the embryonic and juvenile phases of development, and also during spinal cord regeneration. Structurally, the axolotl orthologue of NPDC-1 retained sequence identity to mammalian sequences in all functional domains. Functionally, we observed that axolotl NPDC-1 mRNA expression peaked late in embryogenesis, with highest levels of expression occurring during the time of limb development, a process regulated by retinoic acid signaling. Also similar to what has been observed in mammals, axolotl NPDC-1 directly interacts with axolotl RAR, modulates axolotl RAR DNA binding, and represses cell proliferation and axolotl RAR-mediated gene transcription. These data justify axolotl as a model to further investigate NPDC-1 and its role in regulating retinoic acid signaling. PMID:17331771

Mitochondrial and Chloroplast Stress Responses Are Modulated in Distinct Touch and Chemical Inhibition Phases1[OPEN

PubMed Central

Ivanova, Aneta; Millar, A. Harvey; Whelan, James

2016-01-01

Previous studies have identified a range of transcription factors that modulate retrograde regulation of mitochondrial and chloroplast functions in Arabidopsis (Arabidopsis thaliana). However, the relative importance of these regulators and whether they act downstream of separate or overlapping signaling cascades is still unclear. Here, we demonstrate that multiple stress-related signaling pathways, with distinct kinetic signatures, converge on overlapping gene sets involved in energy organelle function. The transcription factor ANAC017 is almost solely responsible for transcript induction of marker genes around 3 to 6 h after chemical inhibition of organelle function and is a key regulator of mitochondrial and specific types of chloroplast retrograde signaling. However, an independent and highly transient gene expression phase, initiated within 10 to 30 min after treatment, also targets energy organelle functions, and is related to touch and wounding responses. Metabolite analysis demonstrates that this early response is concurrent with rapid changes in tricarboxylic acid cycle intermediates and large changes in transcript abundance of genes encoding mitochondrial dicarboxylate carrier proteins. It was further demonstrated that transcription factors AtWRKY15 and AtWRKY40 have repressive regulatory roles in this touch-responsive gene expression. Together, our results show that several regulatory systems can independently affect energy organelle function in response to stress, providing different means to exert operational control. PMID:27208304

Role of the terminator hairpin in the biogenesis of functional Hfq-binding sRNAs

PubMed Central

Morita, Teppei; Nishino, Ryo; Aiba, Hiroji

2017-01-01

Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3′ end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs. PMID:28606943

Crosstalk between virulence loci: regulation of Salmonella enterica pathogenicity island 1 (SPI-1) by products of the std fimbrial operon.

PubMed

López-Garrido, Javier; Casadesús, Josep

2012-01-01

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam(-) background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam(+) background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam(-) mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed.

Corepressors TLE1 and TLE3 interact with HESX1 and PROP1.

PubMed

Carvalho, Luciani R; Brinkmeier, Michelle L; Castinetti, Frederic; Ellsworth, Buffy S; Camper, Sally A

2010-04-01

Pituitary hormone deficiency causes short stature in one in 4000 children born and can be caused by mutations in transcription factor genes, including HESX1, PROP1, and POU1F1. HESX1 interacts with a member of the groucho-related gene family, TLE1, through an engrailed homology domain and represses PROP1 activity. Mice with Prop1 deficiency exhibit failed differentiation of the POU1F1 lineage, resulting in lack of TSH, GH, and prolactin. In addition, these mutants exhibit profound pituitary dysmorphology and excess Hesx1 and Tle3 expression. The ability of HESX1 to interact with TLE3 has not been explored previously. We tested the ability of TLE3 to enhance HESX1-mediated repression of PROP1 in cell culture. Both TLE3 and TLE1 repress PROP1 in conjunction with HESX1 with similar efficiencies. TLE1 and TLE3 can each repress PROP1 in the absence of HESX1 via a protein-protein interaction. We tested the functional consequences of ectopic TLE3 and HESX1 expression in transgenic mice by driving constitutive expression in pituitary thyrotrophs and gonadotrophs. Terminal differentiation of these cells was suppressed by HESX1 alone and by TLE3 and HESX1 together but not by TLE3 alone. In summary, we present evidence that HESX1 is a strong repressor that can be augmented by the corepressors TLE1 and TLE3. Our in vitro studies suggest that TLE1 and TLE3 might also play roles independent of HESX1 by interacting with other transcription factors like PROP1.

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter

NASA Technical Reports Server (NTRS)

Frudakis, T. N.; Wilt, F.

1995-01-01

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

TCF7L1 recruits CtBP and HDAC1 to repress DICKKOPF4 gene expression in human colorectal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eshelman, Melanie A.; Shah, Meera; Raup-Konsavage, Wesley M.

The T-cell factor/Lymphoid enhancer factor (TCF/LEF; hereafter TCF) family of transcription factors are critical regulators of colorectal cancer (CRC) cell growth. Of the four TCF family members, TCF7L1 functions predominantly as a repressor of gene expression. Few studies have addressed the role of TCF7L1 in CRC and only a handful of target genes regulated by this repressor are known. By silencing TCF7L1 expression in HCT116 cells, we show that it promotes cell proliferation and tumorigenesis in vivo by driving cell cycle progression. Microarray analysis of transcripts differentially expressed in control and TCF7L1-silenced CRC cells identified genes that control cell cycle kinetics andmore » cancer pathways. Among these, expression of the Wnt antagonist DICKKOPF4 (DKK4) was upregulated when TCF7L1 levels were reduced. We found that TCF7L1 recruits the C-terminal binding protein (CtBP) and histone deacetylase 1 (HDAC1) to the DKK4 promoter to repress DKK4 gene expression. In the absence of TCF7L1, TCF7L2 and β-catenin occupancy at the DKK4 promoter is stimulated and DKK4 expression is increased. These findings uncover a critical role for TCF7L1 in repressing DKK4 gene expression to promote the oncogenic potential of CRCs. - Highlights: • TCF7L1 promotes colorectal cancer cell proliferation and tumorigenesis. • DICKKOPF4 is directly regulated by TCF7L1. • TCF7L1 recruits CtBP and HDAC1 to repress DKK4 gene expression.« less

Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation

PubMed Central

Dalbiès-Tran, Rozenn; Stigger-Rosser, Evelyn; Dotson, Travis; Sample, Clare E.

2001-01-01

Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor Jκ. This interaction downregulates transcription mediated by EBNA-2 and Jκ. To identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with Jκ. Using a reporter gene assay based on the recruitment of Jκ by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of Jκ to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with Jκ. To determine the biological significance of the interaction of EBNA-3A with Jκ, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with Jκ through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and Jκ. The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes. PMID:11119577

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

PubMed

Cruickshank, Mark N; Dods, James; Taylor, Rhonda L; Karimi, Mahdad; Fenwick, Emily J; Quail, Elizabeth A; Rea, Alexander J; Holers, V Michael; Abraham, Lawrence J; Ulgiati, Daniela

2015-07-01

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

NF-{kappa}B p65 represses {beta}-catenin-activated transcription of cyclin D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

PubMed Central

Caswell, Clayton C.; Elhassanny, Ahmed E. M.; Planchin, Emilie E.; Roux, Christelle M.; Weeks-Gorospe, Jenni N.; Ficht, Thomas A.; Dunman, Paul M.

2013-01-01

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants. PMID:23319565

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling

PubMed Central

Winther, Sine V.; Tuomainen, Tomi; Borup, Rehannah; Tavi, Pasi; Antoons, Gudrun; Thomsen, Morten B.

2016-01-01

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca2+ dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca2+ ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca2+ concentration. Neither increasing nor decreasing intracellular Ca2+ concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2−/− heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca2+. Thus, KChIP2 does not function as a conventional transcription factor in the heart. PMID:27349185

Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

NASA Astrophysics Data System (ADS)

Wang, Yuh-Hwa; Amirhaeri, Sorour; Kang, Seongman; Wells, Robert D.; Griffith, Jack D.

1994-07-01

The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression

PubMed Central

Garcia, Gloria R.; Goodale, Britton C.; Wiley, Michelle W.; La Du, Jane K.; Hendrix, David A.

2017-01-01

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway. PMID:28385905

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression.

PubMed

Garcia, Gloria R; Goodale, Britton C; Wiley, Michelle W; La Du, Jane K; Hendrix, David A; Tanguay, Robert L

2017-06-01

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA ( slincR ) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway. Copyright © 2017 by The Author(s).