H2A ubiquitination is essential for Polycomb Repressive Complex 1-mediated gene regulation in Marchantia polymorpha
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ABSTRACT: Polycomb repressive complex 1 (PRC1) and PRC2 are chromatin regulators maintaining transcriptional repression by depositing H2A mono-ubiquitination (H2Aub) or H3 lysine 27 tri-methylation (H3K27me3), respectively. While PRC1 is known to be required for transcriptional repression, the contribution of H2Aub in the Polycomb repressive system remains unclear in plants. Here, we directly test the requirement of H2Aub for gene repression in Marchantia polymorpha by generating point mutants in H2A that fail to be ubiquitinated by PRC1. These mutants show reduced H3K27me3 levels on the same target sites as mutants in PRC1 subunits MpBMI1 and the homolog MpBMI1L, revealing that PRC1-catalyzed H2Aub is essential for Polycomb system function. Furthermore, we demonstrate that H2Aub contributes to the PRC1-mediated silencing of genes and transposable elements. Together, our data provide evidence that H2Aub plays indispensable roles in the PRC1-initiated repressive system in Marchantia.
Project description:• Polycomb (PcG) regulation is crucial for development across eukaryotes, but how PcG targets are specified is still incompletely understood. The slow timescale of cold-induced Polycomb Repressive Complex 2 silencing during vernalization at Arabidopsis thaliana FLOWERING LOCUS C (FLC) provides an excellent system to elucidate the sequence of events. Association of the DNA binding protein VAL1 to an FLC intronic RY motif within the PcG nucleation region is an important step. VAL1 is associated in vivo with APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and Polycomb Repressive Complex 1 (PRC1). Here, we show that ASAP and PRC1 functions are necessary for co-transcriptional repression and chromatin regulation during FLC silencing. ASAP mutants affect FLC transcription in warm conditions, but the rate of FLC silencing in the cold is unaffected. PRC1-mediated H2Aub accumulation increases at the nucleation region upon exposure to cold, but unlike the PRC2-delivered H3K27me3 does not spread across the locus. H2Aub is thus involved in the transition to epigenetic silencing at FLC facilitating H3K27me3 accumulation, which in turn is necessary for long-term epigenetic memory. Overall, our work highlights the importance of the DNA sequence-specific binding protein VAL1 as an assembly platform coordinating the co-transcriptional repression and chromatin regulation necessary for the epigenetic silencing of FLC.
Project description:Polycomb (PcG) silencing is crucial for development, but how targets are specified remains incompletely understood. The cold-induced Polycomb Repressive Complex 2 (PRC2) silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC) provides an excellent system to elucidate PcG regulation. Association of the DNA binding protein VAL1 to the PcG nucleationregion at FLC is an important step. VAL1 interacts with APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and PRC1. Here, we show that ASAP and PRC1 are necessary for co-transcriptional repression and chromatin regulation during FLC silencing. ASAP mutants affect FLC transcription in warm conditions, but the rate of FLC silencing in the cold is unaffected. PRC1-mediated H2Aub accumulates at FLC nucleation region during cold, but unlike the PRC2-delivered H3K27me3 does not spread across the locus. H2Aub thus marks the transition to epigenetic silencing, while H3K27me3 is necessary for long-term epigenetic memory. Overall, our work highlights the importance of VAL1 as an assembly platform co-ordinating the co-transcriptional repression and chromatin regulation necessary for the epigenetic silencing of FLC.
Project description:Polycomb group (PcG) proteins play critical roles during development. H2Aub and H3K27me3 are deposited by Polycomb-repressive Complex 1 and 2 (PRC1/2) respectively, and largely overlap in the genome due to mutual recruitment of the two complexes. However, whether PRC1/H2Aub and PRC2/H3K27me3 also function independently remains elusive. Here we uncovered a large-scale decoupling of H2Aub and H3K27me3 in preimplantation mouse embryos, at both canonical PcG targets and broad distal domains. H2Aub represses future bivalent genes without H3K27me3 but does not contribute to maintenance of H3K27me3-dependent non-canonical imprinting. Our study thus revealed their distinct and independent functions in early mammalian development.
Project description:The Polycomb repression machinery in Drosophila comprises PRC1 that monoubiquitinates histone H2A at lysine 118 (H2Aub1) and PR-DUB, a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains enigmatic. We show that H2Aub1 is enriched at Polycomb target genes in early embryos but depleted from these genes during developmental stages when PRC1 represses their transcription. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high-level H2Aub1 accumulation at Polycomb targets increases chromatin accessibility, consistent with disruption of chromatin fiber folding by H2Aub1 in vitro. Consequently, PR-DUB mutants show defective Polycomb repression, while general transcription is largely unperturbed by the genome-wide, low-level H2Aub1 increase. Changes in H2Aub1 levels alter H3K27 methylation-kinetics but PRC2 nevertheless generates canonical H3K27me3 domains in PRC1 or PR-DUB catalytic mutants. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, though deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.
Project description:The Polycomb repression machinery in Drosophila comprises PRC1 that monoubiquitinates histone H2A at lysine 118 (H2Aub1) and PR-DUB, a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains enigmatic. We show that H2Aub1 is enriched at Polycomb target genes in early embryos but depleted from these genes during developmental stages when PRC1 represses their transcription. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high-level H2Aub1 accumulation at Polycomb targets increases chromatin accessibility, consistent with disruption of chromatin fiber folding by H2Aub1 in vitro. Consequently, PR-DUB mutants show defective Polycomb repression, while general transcription is largely unperturbed by the genome-wide, low-level H2Aub1 increase. Changes in H2Aub1 levels alter H3K27 methylation-kinetics but PRC2 nevertheless generates canonical H3K27me3 domains in PRC1 or PR-DUB catalytic mutants. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, though deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.
Project description:The Polycomb repression machinery in Drosophila comprises PRC1 that monoubiquitinates histone H2A at lysine 118 (H2Aub1) and PR-DUB, a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains enigmatic. We show that H2Aub1 is enriched at Polycomb target genes in early embryos but depleted from these genes during developmental stages when PRC1 represses their transcription. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high-level H2Aub1 accumulation at Polycomb targets increases chromatin accessibility, consistent with disruption of chromatin fiber folding by H2Aub1 in vitro. Consequently, PR-DUB mutants show defective Polycomb repression, while general transcription is largely unperturbed by the genome-wide, low-level H2Aub1 increase. Changes in H2Aub1 levels alter H3K27 methylation-kinetics but PRC2 nevertheless generates canonical H3K27me3 domains in PRC1 or PR-DUB catalytic mutants. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, though deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.
Project description: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.
Project description:Polycomb Repressive Complexes 1 and 2 (PRC1 and 2) play a critical role in the epigenetic regulation of transcription during cellular differentiation, stem cell pluripotency, and neoplastic progression1-3. Here we show that the Polycomb group protein EED, a core component of PRC2, physically interacts with and functions as part of the PRC1 complex. Components of PRC1 and PRC2 compete for EED binding. EED functions to recruit PRC1 to H3K27me3 loci and enhances PRC1 mediated H2A ubiquitin E3 ligase activity. Taken together, we uncover the integral role of EED as an epigenetic exchange factor coordinating the activities of PRC1 and 2. EED, uH2A, RING1A, RING1B, BMI1 and H3K27Me3 ChIP-seq in EED stable knockdown and control Scramble DU145 prostate cancer cell line
Project description:Maged1 inactivation induces insensitivity to cocaine by impairing cocaine-evoked release of dopamine and plasticity in the Nucleus accumbens (NAc). Here we have characterized the chromatin epigenetic and transcriptional alterations associated with chronic cocaine exposure in the paraventricular thalamus (PVT, one of the main regulatory inputs of the Nac) in mice WT or conditional KO for Maged1 activity. At the epigenetic level, we focused on the analysis of the genomic coverages of the histone post-translational modification H2AK119ub (monoubiquitination of Lysine 119 of H2A; H2Aub), a repressive chromatin mark associated to polycomb (PRC1) –mediated gene repression since Maged1 specifically interact with H2A upon cocaine administration and is required for cocaine-induced increase of H2AK119ub levels in the PVT.
Project description: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. In contrast to prevailing views, 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.