The Polycomb system modifies chromatin and plays an essential role in repressing gene expression to control normal mammalian development. However, the components and mechanisms that define how ...Polycomb protein complexes achieve this remain enigmatic. Here, we use combinatorial genetic perturbation coupled with quantitative genomics to discover the central determinants of Polycomb-mediated gene repression in mouse embryonic stem cells. We demonstrate that canonical Polycomb repressive complex 1 (PRC1), which mediates higher-order chromatin structures, contributes little to gene repression. Instead, we uncover an unexpectedly high degree of synergy between variant PRC1 complexes, which is fundamental to gene repression. We further demonstrate that variant PRC1 complexes are responsible for distinct pools of H2A monoubiquitylation that are associated with repression of Polycomb target genes and silencing during X chromosome inactivation. Together, these discoveries reveal a new variant PRC1-dependent logic for Polycomb-mediated gene repression.
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•Canonical PRC1 complexes contribute little to H2AK119ub1 and gene repression•Variant PRC1 complexes deposit H2AK119ub1 broadly throughout the genome•Pervasive deposition of H2AK119ub1 by PCGF3/5-PRC1 is linked to X chromosome silencing•Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression
In this article, Fursova et al. uncover the central determinants of Polycomb-mediated gene repression in ESCs. They demonstrate that deposition of H2AK119ub1 and gene repression is driven by synergy between variant PRC1 complexes with little contribution from canonical PRC1 complexes, which mediate higher-order chromatin structures.
Recruitment of the Polycomb repressive complexes PRC1 and PRC2 by Xist RNA is an important paradigm for chromatin regulation by long noncoding RNAs. Here, we show that the noncanonical Polycomb group ...RING finger 3/5 (PCGF3/5)–PRC1 complex initiates recruitment of both PRC1 and PRC2 in response to Xist RNA expression. PCGF3/5–PRC1–mediated ubiquitylation of histone H2A signals recruitment of other noncanonical PRC1 complexes and of PRC2, the latter leading to deposition of histone H3 lysine 27 methylation chromosome-wide. Pcgf3/5 gene knockout results in female-specific embryo lethality and abrogates Xist-mediated gene repression, highlighting a key role for Polycomb in Xist-dependent chromosome silencing. Our findings overturn existing models for Polycomb recruitment by Xist RNA and establish precedence for H2AK119μ1 in initiating Polycomb domain formation in a physiological context.
Abstract
Polycomb repressive complexes-1 and -2 (PRC1 and 2) silence developmental genes in a spatiotemporal manner during embryogenesis. How Polycomb group (PcG) proteins orchestrate down-regulation ...of target genes upon differentiation, however, remains elusive. Here, by differentiating embryonic stem cells into embryoid bodies, we reveal a crucial role for the PCGF1-containing variant PRC1 complex (PCGF1-PRC1) to mediate differentiation-associated down-regulation of a group of genes. Upon differentiation cues, transcription is down-regulated at these genes, in association with PCGF1-PRC1-mediated deposition of histone H2AK119 mono-ubiquitination (H2AK119ub1) and PRC2 recruitment. In the absence of PCGF1-PRC1, both H2AK119ub1 deposition and PRC2 recruitment are disrupted, leading to aberrant expression of target genes. PCGF1-PRC1 is, therefore, required for initiation and consolidation of PcG-mediated gene repression during differentiation.
Polycomb repressive complex (PRC) 1 maintains developmental genes in a poised state through monoubiquitination (Ub) of histone H2A. Although Ub-independent functions of PRC1 have also been suggested, ...it has remained unclear whether Ub-dependent and -independent functions of PRC1 operate differentially in a developmental context. Here, we show that the E3 ubiquitin ligase activity of Ring1B, a core component of PRC1, is necessary for the temporary repression of key neuronal genes in neurogenic (early-stage) neural stem or progenitor cells (NPCs) but is dispensable for the persistent repression of these genes associated with the loss of neurogenic potential in astrogliogenic (late-stage) NPCs. Our results also suggest that histone deacetylase (HDAC) activity of the NuRD/MBD3 complex and Phc2-dependent PRC1 clustering are necessary for the transition from the Ub-dependent to -independent function of PRC1. Together, these results indicate that Ub-independent mode of repression by PRC1 plays a key role in mammalian development during cell fate restriction.
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•A bona fide Ub(−) Ring1 mutant proves its Ub-independent role in mammalian development•Neuronal genes are rendered ‘poised’ in a Ub-dependent manner in neurogenic NPCs•These genes are persistently repressed independently of Ub in non-neurogenic NPCs•Histone deacetylation and Phc2 clustering mediate PRC1’s Ub-independent repression mode
Histone H2A ubiquitination (Ub)-dependent and -independent roles have been suggested for PRC1-Ring1. Tsuboi et al. show that PRC1 relies on Ub for transient silencing of poised neuronal genes in neurogenic neural stem/progenitor cells and persistently silences the same genes independently of Ub during neuronal fate restriction in mammalian development.
Silencing of a subset of germline genes is dependent upon DNA methylation (DNAme) post-implantation. However, these genes are generally hypomethylated in the blastocyst, implicating alternative ...repressive pathways before implantation. Indeed, in embryonic stem cells (ESCs), an overlapping set of genes, including germline "genome-defence" (GGD) genes, are upregulated following deletion of the H3K9 methyltransferase SETDB1 or subunits of the non-canonical PRC1 complex PRC1.6. Here, we show that in pre-implantation embryos and naïve ESCs (nESCs), hypomethylated promoters of germline genes bound by the PRC1.6 DNA-binding subunits MGA/MAX/E2F6 are enriched for RING1B-dependent H2AK119ub1 and H3K9me3. Accordingly, repression of these genes in nESCs shows a greater dependence on PRC1.6 than DNAme. In contrast, GGD genes are hypermethylated in epiblast-like cells (EpiLCs) and their silencing is dependent upon SETDB1, PRC1.6/RING1B and DNAme, with H3K9me3 and DNAme establishment dependent upon MGA binding. Thus, GGD genes are initially repressed by PRC1.6, with DNAme subsequently engaged in post-implantation embryos.
Epigenetic inheritance of DNA methylation in mammals requires a multifunctional protein UHRF1, which is believed to recruit DNMT1 to DNA replication forks through a unique hemi-methylated CpG-binding ...activity. Here we demonstrate that the UHRF1 mutants deficient in binding either hemi-methylated CpG or H3K9me2/3, but not both, are able to associate with pericentric heterochromatin, recruit Dnmt1 and partially rescue DNA methylation defects in mouse Uhrf1 null ES cells. Furthermore, we present evidence that the flip out of the methylated cytosine induced by UHRF1 binding is unlikely essential for subsequent DNA methylation by DNMT1. Together, our study demonstrates that UHRF1 can target DNMT1 for DNA maintenance methylation through binding either H3K9me2/3 or hemi-methylated CpG, and that the presence of both binding activities ensures high fidelity DNA maintenance methylation. In addition, our study indicates that UHRF1 mediates cross-talk between H3K9 methylation and DNA methylation at the level of DNA methylation maintenance.
CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate ...expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs.DOI:http://dx.doi.org/10.7554/eLife.00205.001.
Hemimethylation: DNA's lasting odd couple Sharif, Jafar; Koseki, Haruhiko
Science (American Association for the Advancement of Science),
03/2018, Letnik:
359, Številka:
6380
Journal Article
Recenzirano
Stably inherited DNA hemimethylation regulates chromatin interaction and transcription
DNA methylation is an essential epigenetic modification that regulates gene transcription, embryonic ...development, and cell differentiation in both animals and plants. In mammals, DNA methylation generally occurs at CpG dinucleotides in a symmetric fashion (
1
), meaning that if a cytosine (C) residue on one CpG is methylated, the corresponding residue on the complementary strand will be too. This pattern temporarily breaks down during DNA replication, when the unmethylated daughter (nascent) strand and the methylated parent strand create an asymmetrically methylated CpG dyad termed hemimethylated DNA. It was thought that the destiny of hemimethylated DNA was to become fully methylated or unmethylated by replication-coupled dilution. However, about 10% of CpGs in embryonic stem cells (ESCs) (
1
) and trophoblast stem cells (
2
) remain hemimethylated. It is not known if this unusual hemimethylation signature occurs by chance or by design. On page 1166 of this issue, Xu and Corces (
3
) reveal that some CpGs in the genome can be hemimethylated by design. Intriguingly, they found that hemimethylated sites are inherited over several cell divisions. This challenges the prevailing view that hemimethylation is transient and suggests that this DNA modification could be maintained as a stable epigenetic state.
The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of ...p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a “recruit-and-release” mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.
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•p300/CBP and deacetylase activities regulate dynamic (de)activation of enhancers•p300/CBP-catalyzed acetylation promotes PIC assembly and RNAPII recruitment•BRD4 acts as a p300/CBP downstream effector to promote RNAPII pause release•Coupling of RNAPII recruitment and pause release enables rapid enhancer activation
A systems-wide analysis reveals that enhancers are activated by p300/CBP-catalyzed acetylation and deactivated by deacetylation. p300/CBP activity promotes pre-initiation complex assembly and RNAPII recruitment independently of its previously known function in BRD4-dependent pause release. By simultaneously promoting transcription initiation and elongation, p300/CBP activity drives the rapid activation of enhancers and super-enhancers.
The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic ...male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.
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