Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression

SummaryStress-induced readthrough transcription results in the synthesis of thousands of downstream-of-gene (DoG) containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse-seq revealed that hyperosmotic stress induces widespread transcriptional repression. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP-seq confirmed that the stress-induced redistribution of RNA Polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While subunits of the cleavage and polyadenylation machinery remained Pol II-associated, Integrator complex subunits dissociated from Pol II under stress conditions. Depleting the catalytic subunit of the Integrator complex, Int11, using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.In briefRosa-Mercado et al. report that hyperosmotic stress causes widespread transcriptional repression in human cells, yet DoGs arise regardless of the transcriptional response of their upstream genes. They find that the interaction between Pol II and Integrator is disrupted by hypertonicity and that knocking down the Integrator nuclease leads to DoG production.HighlightsHyperosmotic stress triggers transcriptional repression of many genes.DoG RNAs arise independent of the transcriptional level of their upstream gene.The interaction between Pol II and Integrator subunits decreases after salt stress.Depletion of the Int11 nuclease subunit induces the production of hundreds of DoGs.

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Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.2970-2983.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 2970-2983 ◽

Cited By ~ 141

Author(s):

Dmitri Ivanov ◽

Youn Tae Kwak ◽

Jun Guo ◽

Richard B. Gaynor

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Transcriptional Elongation ◽

Tat Protein ◽

C Terminus ◽

Heptad Repeats ◽

And Function ◽

Hiv 1

ABSTRACT SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.

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PPARβ/δ controls Mediator recruitment and transcription initiation

10.1101/525576 ◽

2019 ◽

Author(s):

Nathalie Legrand ◽

Clemens L. Bretscher ◽

Svenja Zielke ◽

Bernhard Wilke ◽

Michael Daude ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Transcription Initiation ◽

Target Genes ◽

Hdac Inhibitors ◽

Inverse Agonist ◽

Inverse Agonists ◽

A Subunit ◽

Basal Transcription Factors

AbstractRepression of transcription by nuclear receptors involves NCOR and SMRT corepressor complexes, which harbour the deacetylase HDAC3 as a subunit. Both deacetylase-dependent and -independent repression mechanisms have been reported for these complexes. In the absence of ligands, the nuclear receptor PPARβ/δ recruits NCOR and SMRT and represses expression of its canonical targets including the ANGPTL4 gene. Agonistic ligands cause corepressor dissociation and enable enhanced induction of transcription by coactivators. Vice versa, recently developed synthetic inverse agonists lead to augmented corepressor recruitment and repression that dominates over activating stimuli. Both basal repression of ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question of how PPARβ/δ represses transcription mechanistically.Here, we show that the PPARβ/δ inverse agonist PT-S264 impairs transcription initiation in human cells. Inverse agonist-bound PPARβ/δ interferes with recruitment of Mediator, RNA polymerase II, and TFIIB, but not with recruitment of other basal transcription factors, to the ANGPTL4 promoter. We identify NCOR as the main ligand-dependent interactor of PPARβ/δ in the presence of PT-S264. In PPARβ/δ knockout cells, reconstitution with PPARβ/δ mutants deficient in basal repression recruit less NCOR, SMRT, and HDAC3 to chromatin, concomitant with increased binding of RNA polymerase II. PT-S264 restores binding of NCOR, SMRT, and HDAC3, resulting in diminished polymerase II binding and transcriptional repression. In the presence of HDAC inhibitors, ligand-mediated repression of PPARβ/δ target genes is only partially relieved. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes. Deacetylase-independent repression mediated by binding of inverse agonists to PPARβ/δ involve NCOR/SMRT recruitment and interference with Mediator, TFIIB, and RNA polymerase II binding.

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Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins

The Journal of Cell Biology ◽

10.1083/jcb.200407182 ◽

2004 ◽

Vol 168 (1) ◽

pp. 41-54 ◽

Cited By ~ 147

Author(s):

Danyang Chen ◽

Miroslav Dundr ◽

Chen Wang ◽

Anthony Leung ◽

Angus Lamond ◽

...

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Core Histone ◽

Mitotic Chromosomes ◽

Chromatin Structural ◽

Polymerase I ◽

Kinetics Of ◽

Mitotic Chromatin

During mitosis, chromosomes are highly condensed and transcription is silenced globally. One explanation for transcriptional repression is the reduced accessibility of transcription factors. To directly test this hypothesis and to investigate the dynamics of mitotic chromatin, we evaluate the exchange kinetics of several RNA polymerase I transcription factors and nucleosome components on mitotic chromatin in living cells. We demonstrate that these factors rapidly exchange on and off ribosomal DNA clusters and that the kinetics of exchange varies at different phases of mitosis. In addition, the nucleosome component H1c-GFP also shows phase-specific exchange rates with mitotic chromatin. Furthermore, core histone components exchange at detectable levels that are elevated during anaphase and telophase, temporally correlating with H3-K9 acetylation and recruitment of RNA polymerase II before the onset of bulk RNA synthesis at mitotic exit. Our findings indicate that mitotic chromosomes in general and ribosomal genes in particular, although highly condensed, are accessible to transcription factors and chromatin proteins. The phase-specific exchanges of nucleosome components during late mitotic phases are consistent with an emerging model of replication independent core histone replacement.

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Transcriptional Repression of Tumor SuppressorCDC73, Encoding an RNA Polymerase II Interactor, by Wilms Tumor 1 Protein (WT1) Promotes Cell Proliferation

Journal of Biological Chemistry ◽

10.1074/jbc.m113.483255 ◽

2013 ◽

Vol 289 (2) ◽

pp. 968-976 ◽

Cited By ~ 16

Author(s):

Mohammad Iqbal Rather ◽

Shivananda Swamy ◽

Kodaganur S. Gopinath ◽

Arun Kumar

Keyword(s):

Cell Proliferation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Wilms Tumor ◽

Wilms Tumor 1

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Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination

Molecular and Cellular Biology ◽

10.1128/mcb.00181-18 ◽

2018 ◽

Vol 38 (18) ◽

Cited By ~ 7

Author(s):

Joseph F. Cardiello ◽

James A. Goodrich ◽

Jennifer F. Kugel

Keyword(s):

Heat Shock ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Normal Growth ◽

Global Changes ◽

Transcriptional Initiation ◽

Pol Ii ◽

Transcriptional Termination ◽

Number Of Genes

ABSTRACT Cellular transcriptional programs are tightly controlled but can profoundly change in response to environmental challenges or stress. Here we describe global changes in mammalian RNA polymerase II (Pol II) occupancy at mRNA genes in response to heat shock and after recovery from the stress. After a short heat shock, Pol II occupancy across thousands of genes decreased, consistent with widespread transcriptional repression, whereas Pol II occupancy increased at a small number of genes in a manner consistent with activation. Most striking, however, was loss of the Pol II peak near the 3′ ends of mRNA genes, coupled to a gain in polymerase occupancy extending tens of kilobases downstream of 3′ ends. Typical patterns of 3′ end occupancy were largely restored 60 min after cells returned to normal growth temperatures. These changes in polymerase occupancy revealed a heat shock-induced loss of normal termination, which was potent, global, and reversible. The occupancy of the termination factor CPSF73 at the 3′ ends of representative genes was reduced after heat shock, suggesting a mechanism for impaired termination. The data support a model in which heat shock induces widespread repression of transcriptional initiation and loss of transcription termination, which reverses as cells return to homeostasis.

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TFIIF Facilitates Dissociation of RNA Polymerase II from Noncoding RNAs That Lack a Repression Domain

Molecular and Cellular Biology ◽

10.1128/mcb.01115-09 ◽

2009 ◽

Vol 30 (1) ◽

pp. 91-97 ◽

Cited By ~ 13

Author(s):

Stacey D. Wagner ◽

Jennifer F. Kugel ◽

James A. Goodrich

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Noncoding Rnas ◽

Mrna Transcription ◽

Rna Pol Ii ◽

Pol Ii ◽

General Transcription Factors ◽

B2 Rna ◽

Repression Domain

ABSTRACT Noncoding RNAs (ncRNAs) have recently been found to regulate multiple steps in mammalian mRNA transcription. Mouse B2 RNA and human Alu RNA bind RNA polymerase II (Pol II) and repress mRNA transcription, using regions of the ncRNAs referred to as repression domains. Two other ncRNAs, mouse B1 RNA and human small cytoplasmic Alu (scAlu) RNA, bind Pol II with high affinity but lack repression domains and hence do not inhibit transcription. To better understand the interplay between ncRNAs that bind Pol II and their functions in transcription, we studied how Pol II binding and transcriptional repression are controlled by general transcription factors. We found that TFIIF associates with B1 RNA/Pol II and scAlu RNA/Pol II complexes and decreases their kinetic stability. Both subunits of TFIIF are required for this activity. Importantly, fusing a repression domain to B1 RNA stabilizes its interaction with Pol II in the presence of TFIIF. These results suggest a new role for TFIIF in regulating the interaction of ncRNAs with Pol II; specifically, it destabilizes interactions with ncRNAs that are not transcriptional repressors. These studies also identify a new function for ncRNA repression domains: they stabilize interactions of ncRNAs with Pol II in the presence of TFIIF.

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Alleviation of Human Papillomavirus E2-Mediated Transcriptional Repression via Formation of a TATA Binding Protein (or TFIID)-TFIIB-RNA Polymerase II-TFIIF Preinitiation Complex

Molecular and Cellular Biology ◽

10.1128/mcb.20.1.113-125.2000 ◽

2000 ◽

Vol 20 (1) ◽

pp. 113-125 ◽

Cited By ~ 38

Author(s):

Samuel Y. Hou ◽

Shwu-Yuan Wu ◽

Tianyuan Zhou ◽

Mary C. Thomas ◽

Cheng-Ming Chiang

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Binding Protein ◽

Viral Gene Expression ◽

Tata Binding Protein ◽

Human Papillomaviruses ◽

Viral Gene ◽

Preinitiation Complex ◽

Hpv E6

ABSTRACT Transcription in human papillomaviruses (HPVs) is mainly regulated by cellular transcription factors and virus-encoded E2 proteins that act as sequence-specific DNA-binding proteins. Although the functions of E2 as a transcriptional activator and a repressor have been well documented, the role of cellular factors involved in E2-mediated regulation of the HPV promoters and the mechanism by which E2 modulates viral gene expression remain unclear. Using reconstituted cell-free transcription systems, we found that cellular enhancer-binding factors and general cofactors, such as TAFIIs, TFIIA, Mediator, and PC4, are not required for E2-mediated repression. Unlike other transcriptional repressors that function through recruitment of histone deacetylase or corepressor complexes, HPV E2 is able to directly target components of the general transcription machinery to exert its repressor activity on the natural HPV E6 promoter. Interestingly, preincubation of TATA binding protein (TBP) or TFIID with HPV template is not sufficient to overcome E2-mediated repression, which can be alleviated only via formation of a minimal TBP (or TFIID)-TFIIB-RNA polymerase II-TFIIF preinitiation complex. Our data therefore indicate that E2 does not simply work by displacing TBP or TFIID from binding to the adjacent TATA box. Instead, E2 appears to function as an active repressor that directly inhibits HPV transcription at steps after TATA recognition by TBP or TFIID.

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Functional Relationships of Srb10-Srb11 Kinase, Carboxy-Terminal Domain Kinase CTDK-I, and Transcriptional Corepressor Ssn6-Tup1

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1163 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1163-1171 ◽

Cited By ~ 82

Author(s):

Sergei Kuchin ◽

Marian Carlson

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Functional Link ◽

Synthetic Promoters ◽

Functional Relationships ◽

Terminal Domain ◽

Carboxy Terminal Domain ◽

Carboxy Terminal ◽

Rna Polymerase Ii Holoenzyme

ABSTRACT The Srb10-Srb11 protein kinase of Saccharomyces cerevisiae is a cyclin-dependent kinase (cdk)-cyclin pair which has been found associated with the carboxy-terminal domain (CTD) of RNA polymerase II holoenzyme forms. Previous genetic findings implicated the Srb10-Srb11 kinase in transcriptional repression. Here we use synthetic promoters and LexA fusion proteins to test the requirement for Srb10-Srb11 in repression by Ssn6-Tup1, a global corepressor. We show that srb10Δ and srb11Δ mutations reduce repression by DNA-bound LexA-Ssn6 and LexA-Tup1. A point mutation in a conserved subdomain of the kinase similarly reduced repression, indicating that the catalytic activity is required. These findings establish a functional link between Ssn6-Tup1 and the Srb10-Srb11 kinase in vivo. We also explored the relationship between Srb10-Srb11 and CTD kinase I (CTDK-I), another member of the cdk-cyclin family that has been implicated in CTD phosphorylation. We show that mutation of CTK1, encoding the cdk subunit, causes defects in transcriptional repression by LexA-Tup1 and in transcriptional activation. Analysis of the mutant phenotypes and the genetic interactions of srb10Δ and ctk1Δ suggests that the two kinases have related but distinct roles in transcriptional control. These genetic findings, together with previous biochemical evidence, suggest that one mechanism of repression by Ssn6-Tup1 involves functional interaction with RNA polymerase II holoenzyme.

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AluMobile Elements: From Junk DNA to Genomic Gems

Scientifica ◽

10.6064/2012/545328 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 8

Author(s):

Sami Dridi

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Genetic Diseases ◽

Rna Polymerase Iii ◽

Mrna Processing ◽

Biological Functions ◽

Junk Dna ◽

Human Genomes ◽

Regulatory Functions

Alus, the short interspersed repeated sequences (SINEs), are retrotransposons that litter the human genomes and have long been considered junk DNA. However, recent findings that these mobile elements are transcribed, both as distinct RNA polymerase III transcripts and as a part of RNA polymerase II transcripts, suggest biological functions and refute the notion thatAlusare biologically unimportant. Indeed,AluRNAs have been shown to control mRNA processing at several levels, to have complex regulatory functions such as transcriptional repression and modulating alternative splicing and to cause a host of human genetic diseases.AluRNAs embedded in Pol II transcripts can promote evolution and proteome diversity, which further indicates that these mobile retroelements are in fact genomic gems rather than genomic junks.

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