PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency

AbstractThe Polycomb repressive system plays a fundamental role in controlling gene expression during mammalian development. To achieve this, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) bind target genes and use histone modification-dependent feedback mechanisms to form Polycomb chromatin domains and repress transcription. The interrelatedness of PRC1 and PRC2 activity at these sites has made it difficult to discover the specific components of Polycomb chromatin domains that drive gene repression and to understand mechanistically how this is achieved. Here, by exploiting rapid degron-based approaches and time-resolved genomics we kinetically dissect Polycomb-mediated repression and discover that PRC1 functions independently of PRC2 to counteract RNA polymerase II binding and transcription initiation. Using single-cell gene expression analysis, we reveal that PRC1 acts uniformly within the cell population, and that repression is achieved by controlling transcriptional burst frequency. These important new discoveries provide a mechanistic and conceptual framework for Polycomb-dependent transcriptional control.

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Stochastic transcription in the p53-mediated response to DNA damage is modulated by burst frequency

10.1101/679449 ◽

2019 ◽

Author(s):

Dhana Friedrich ◽

Laura Friedel ◽

Andreas Herrmann ◽

Stephan Preibisch ◽

Alexander Loewer

Keyword(s):

Gene Expression ◽

Target Genes ◽

Burst Frequency ◽

Time Resolved ◽

Mammalian Cell Lines ◽

Lysine Residues ◽

Acetylation State ◽

Specific Regulation ◽

Discontinuous Transcription ◽

Target Promoters

ABSTRACTDiscontinuous transcription has been described for different mammalian cell lines and numerous promoters. However, our knowledge of how the activity of individual promoters is adjusted by dynamic signaling inputs from transcription factor is limited. To address this question, we characterized the activity of selected target genes that are regulated by pulsatile accumulation of the tumor suppressor p53 in response to ionizing radiation. We performed time resolved measurements of gene expression at the single cell level by smFISH and used the resulting data to inform a mathematical model of promoter activity. We found that p53 target promoters are regulated by frequency modulation of stochastic bursting and can be grouped along three archetypes of gene expression. The occurrence of these archetypes cannot solely be explained by nuclear p53 abundance or promoter binding of total p53. Instead, we provide evidence that the time-varying acetylation state of p53’s C-terminal lysine residues is critical for gene-specific regulation of stochastic bursting.

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Nutrient-regulated gene expression in eukaryotes

Biochemical Society Symposium ◽

10.1042/bss0730085 ◽

2006 ◽

Vol 73 ◽

pp. 85-96 ◽

Cited By ~ 8

Author(s):

Richard J. Reece ◽

Laila Beynon ◽

Stacey Holden ◽

Amanda D. Hughes ◽

Karine Rébora ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Control ◽

Direct Interaction ◽

Signalling Pathways ◽

A Cell ◽

One Step ◽

Higher Eukaryotes ◽

Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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Polycomb Assemblies Multitask to Regulate Transcription

Epigenomes ◽

10.3390/epigenomes3020012 ◽

2019 ◽

Vol 3 (2) ◽

pp. 12 ◽

Cited By ~ 2

Author(s):

Miguel Vidal

Keyword(s):

Gene Expression ◽

Drosophila Melanogaster ◽

Transcriptional Control ◽

Catalytic Properties ◽

Integrated Approach ◽

Gene Repression ◽

Developmental Studies ◽

Topological Domains ◽

Developmental Transitions ◽

New Findings

The Polycomb system is made of an evolutionary ancient group of proteins, present throughout plants and animals. Known initially from developmental studies with the fly Drosophila melanogaster, they were associated with stable sustainment of gene repression and maintenance of cell identity. Acting as multiprotein assemblies with an ability to modify chromatin, through chemical additions to histones and organization of topological domains, they have been involved subsequently in control of developmental transitions and in cell homeostasis. Recent work has unveiled an association of Polycomb components with transcriptionally active loci and the promotion of gene expression, in clear contrast with conventional recognition as repressors. Focusing on mammalian models, I review here advances concerning roles in transcriptional control. Among new findings highlighted is the regulation of their catalytic properties, recruiting to targets, and activities in chromatin organization and compartmentalization. The need for a more integrated approach to the study of the Polycomb system, given its fundamental complexity and its adaptation to cell context, is discussed.

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PPARβ/δ recruits NCOR and regulates transcription reinitiation of ANGPTL4

Nucleic Acids Research ◽

10.1093/nar/gkz685 ◽

2019 ◽

Vol 47 (18) ◽

pp. 9573-9591 ◽

Cited By ~ 1

Author(s):

Nathalie Legrand ◽

Clemens L Bretscher ◽

Svenja Zielke ◽

Bernhard Wilke ◽

Michael Daude ◽

...

Keyword(s):

Mass Spectrometry ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Receptor ◽

Transcription Initiation ◽

Target Gene ◽

Target Genes ◽

Hdac Inhibition ◽

Inverse Agonists ◽

Transcription Reinitiation

Abstract In the absence of ligands, the nuclear receptor PPARβ/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARβ/δ represses transcription mechanistically. We show that the PPARβ/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARβ/δ. Reconstitution of knockout cells with PPARβ/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription.

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The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.01828-06 ◽

2007 ◽

Vol 27 (6) ◽

pp. 2059-2073 ◽

Cited By ~ 85

Author(s):

Victoria H. Cowling ◽

Michael D. Cole

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Target Genes ◽

Dependent Manner ◽

Transactivation Domain ◽

Pol Ii ◽

Terminal Domain ◽

Ctd Phosphorylation

ABSTRACT Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc −/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters and stimulates TFIIH binding in an MBII-dependent manner. Expression of the Myc transactivation domain increases CDK mRNA cap methylation, polysome loading, and the rate of translation. We find that some traditional Myc transcriptional target genes are also regulated by this Myc-driven translation mechanism. We propose that Myc transactivation domain-driven RNA Pol II CTD phosphorylation has broad effects on both transcription and mRNA metabolism.

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Trans-tail regulation-mediated suppression of cryptic transcription

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00711-x ◽

2021 ◽

Author(s):

Jungmin Choi ◽

Zae Young Ryoo ◽

Dong-Hyung Cho ◽

Hyun-Shik Lee ◽

Hong-Yeoul Ryu

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Histone Deacetylases ◽

Transcriptional Control ◽

Molecular Techniques ◽

Transcriptional Elongation ◽

Post Translational Modifications ◽

Phosphorylation Pattern ◽

Cryptic Transcription ◽

H3k79 Methylation

AbstractCrosstalk between post-translational modifications of histone proteins influences the regulation of chromatin structure and gene expression. Among such crosstalk pathways, the best-characterized example is H2B monoubiquitination-mediated H3K4 and H3K79 methylation, which is referred to as trans-tail regulation. Although many studies have investigated the fragmentary effects of this pathway on silencing and transcription, its ultimate contribution to transcriptional control has remained unclear. Recent advances in molecular techniques and genomics have, however, revealed that the trans-tail crosstalk is linked to a more diverse cascade of histone modifications and has various functions in cotranscriptional processes. Furthermore, H2B monoubiquitination sequentially facilitates H3K4 dimethylation and histone sumoylation, thereby providing a binding platform for recruiting Set3 complex proteins, including two histone deacetylases, to restrict cryptic transcription from gene bodies. The removal of both ubiquitin and SUMO, small ubiquitin-like modifier, modifications from histones also facilitates a change in the phosphorylation pattern of the RNA polymerase II C-terminal domain that is required for subsequent transcriptional elongation. Therefore, this review describes recent findings regarding trans-tail regulation-driven processes to elaborate on their contribution to maintaining transcriptional fidelity.

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H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions

10.1101/684712 ◽

2019 ◽

Author(s):

Yichao Cai ◽

Ying Zhang ◽

Yan Ping Loh ◽

Jia Qi Tng ◽

Mei Chee Lim ◽

...

Keyword(s):

Gene Expression ◽

Tumor Growth ◽

Target Genes ◽

Gene Repression ◽

Xenograft Tumor ◽

Regulate Gene Expression ◽

Chromatin Interactions ◽

Genomic Regions ◽

Xenograft Tumor Growth ◽

Regulate Gene

AbstractGene repression and silencers are poorly understood. We reasoned that H3K27me3-rich regions (MRRs) of the genome defined from clusters of H3K27me3 peaks may be used to identify silencers that can regulate gene expression via proximity or looping. MRRs were associated with chromatin interactions and interact preferentially with each other. MRR component removal at interaction anchors by CRISPR led to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. The MRR knockout cells also showed changes in phenotype associated with cell identity, and altered xenograft tumor growth. MRR-associated genes and long-range chromatin interactions were susceptible to H3K27me3 depletion. Our results characterized H3K27me3-rich regions and their mechanisms of functioning via looping.

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Generation and timing of graded responses to morphogen gradients

Development ◽

10.1242/dev.199991 ◽

2021 ◽

Vol 148 (24) ◽

Author(s):

Shari Carmon ◽

Felix Jonas ◽

Naama Barkai ◽

Eyal D. Schejter ◽

Ben-Zion Shilo

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Target Genes ◽

Cell Formation ◽

Embryonic Cell ◽

Mrna Accumulation ◽

Temporal Regulation ◽

Morphogen Gradients ◽

Graded Response ◽

Spatio Temporal

ABSTRACT Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.

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Enhancers predominantly regulate gene expression in vivo via transcription initiation

10.1101/844191 ◽

2019 ◽

Author(s):

Martin S. C. Larke ◽

Takayuki Nojima ◽

Jelena Telenius ◽

Jacqueline A. Sharpe ◽

Jacqueline A. Sloane-Stanley ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Genetically Engineered ◽

Control Gene ◽

Transcriptional Initiation ◽

Pol Ii ◽

Rna Molecules ◽

Control Gene Expression

ABSTRACTGene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

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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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