The polycomb group protein PCGF6 mediates germline gene silencing by recruiting histone-modifying proteins to target gene promoters

Polycomb group (PcG) proteins are essential for maintenance of lineage fidelity by coordinating developmental gene expression programs. Polycomb group ring finger 6 (PCGF6) has been previously reported to repress expression of lineage-specific genes, especially germ cell–related genes in mouse embryonic stem cells (ESCs) via the noncanonical polycomb repressive complex PRC1.6. However, the molecular mechanism of this repression remains largely unknown. Here, using RNA-Seq, real-time RT-PCR, immunohistochemistry, immunoprecipitation, and ChIP analyses, we demonstrate that PCGF6 plays an essential role in embryonic development, indicated by the partially penetrant embryonic lethality in homozygous PCGF6 (Pcgf6−/−)-deficient mice. We also found that surviving Pcgf6-deficient mice exhibit reduced fertility. Using the Pcgf6-deficient mice, we observed that ablation of Pcgf6 in somatic tissues robustly derepresses germ cell–related genes. We further provide evidence that these genes are direct targets of PCGF6 in ESCs and that endogenous PCGF6 co-localizes with the histone-modifying proteins G9A histone methyltransferase (G9A)/G9a-like protein (GLP) and histone deacetylase 1/2 (HDAC1/2) on the promoters of the germ cell–related genes. Moreover, the binding of these proteins to their target genes correlated with methylation of Lys-9 of histone 3 and with the status of histone acetylation at these genes. Moreover, the recruitment of G9A/GLP and HDAC1/2 to target promoters depended on the binding of PCGF6. Our findings indicate that PCGF6 has a critical role in safeguarding lineage decisions and in preventing aberrant expression of germ cell–related genes.

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Variant PCGF1-PRC1 links PRC2 recruitment with differentiation-associated transcriptional inactivation at target genes

Nature Communications ◽

10.1038/s41467-021-24894-z ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Hiroki Sugishita ◽

Takashi Kondo ◽

Shinsuke Ito ◽

Manabu Nakayama ◽

Nayuta Yakushiji-Kaminatsui ◽

...

Keyword(s):

Stem Cells ◽

Crucial Role ◽

Target Genes ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Polycomb Group ◽

Gene Repression ◽

Down Regulation ◽

Aberrant Expression ◽

Polycomb Repressive Complexes

AbstractPolycomb 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.

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Polycomb repressive complex 1 defines the nucleosome landscape but not accessibility at target genes

10.1101/280305 ◽

2018 ◽

Author(s):

Hamish W King ◽

Robert J Klose

Keyword(s):

Target Genes ◽

Nucleosome Occupancy ◽

Embryonic Stem ◽

Chromatin Accessibility ◽

Polycomb Group ◽

Gene Promoters ◽

Polycomb Repressive Complex ◽

Chromatin Domains ◽

Genetic Ablation ◽

Polycomb Repressive Complex 1

ABSTRACTPolycomb group (PcG) proteins are transcriptional repressors that play important roles regulating gene expression during animal development. In vitro experiments have shown that PcG protein complexes can compact chromatin limiting the activity of chromatin remodelling enzymes and access of the transcriptional machinery to DNA. In fitting with these ideas, gene promoters associated with PcG proteins have been reported to be less accessible than other gene promotors. However, it remains largely untested in vivo whether PcG proteins define chromatin accessibility or other chromatin features. To address this important question, we measured chromatin accessibility and examined the nucleosome landscape at PcG protein-bound promoters in mouse embryonic stem cells using the assay for transposase accessible chromatin (ATAC)-seq. Combined with genetic ablation strategies, we unexpectedly discover that although PcG protein-occupied gene promoters exhibit reduced accessibility, this does not rely on PcG proteins. Instead, the Polycomb repressive complex 1 (PRC1) appears to play a unique role in driving elevated nucleosome occupancy and decreased nucleosomal spacing in Polycomb chromatin domains. Our new genome-scale observations argue, in contrast to the prevailing view, that PcG proteins and Polycomb chromatin domains do not significantly affect chromatin accessibility and highlight an underappreciated complexity in the relationship between chromatin accessibility, the nucleosome landscape and PcG-mediated transcriptional repression.

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PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes

eLife ◽

10.7554/elife.21064 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

Mitsuhiro Endoh ◽

Takaho A Endo ◽

Jun Shinga ◽

Katsuhiko Hayashi ◽

Anca Farcas ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Germ Cell ◽

Target Genes ◽

Embryonic Stem ◽

Ring Finger ◽

Mouse Embryonic Stem Cells ◽

Polycomb Group ◽

Histone H2a ◽

Cell Growth And Viability

The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells.

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PICOT binding to chromatin-associated EED negatively regulates cyclin D2 expression by increasing H3K27me3 at the CCND2 gene promoter

Cell Death and Disease ◽

10.1038/s41419-019-1935-0 ◽

2019 ◽

Vol 10 (10) ◽

Cited By ~ 2

Author(s):

Pinakin Pandya ◽

Minesh Jethva ◽

Eitan Rubin ◽

Ramon Y. Birnbaum ◽

Alex Braiman ◽

...

Keyword(s):

Target Genes ◽

Patient Survival ◽

Leukemia Cell ◽

Gene Promoter ◽

Human Tumors ◽

Polycomb Group ◽

The Cancer Genome Atlas ◽

Polycomb Group Protein ◽

Cyclin D2 ◽

Human Cancers

Abstract Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Grx3; Glrx3)) is a ubiquitous protein that can interact with the embryonic ectoderm development (EED) protein via each of its two C-terminal PICOT/Grx homology domains. Since EED is a Polycomb-Group protein and a core component of the polycomb repressive complex 2 (PRC2), we tested the involvement of PICOT in the regulation of PRC2-mediated H3 lysine 27 trimethylation (H3K27me3), transcription and translation of selected PRC2 target genes. A fraction of the cellular PICOT protein was found in the nuclei of leukemia cell lines, where it was associated with the chromatin. In addition, PICOT coimmunoprecipitated with chromatin-residing EED derived from Jurkat and COS-7 cell nuclei. PICOT knockdown led to a reduced H3K27me3 mark and a decrease in EED and EZH2 at the CCND2 gene promoter. In agreement, PICOT-deficient T cells exhibited a significant increase in CCND2 mRNA and protein expression. Since elevated expression levels of PICOT were reported in several different tumors and correlated in the current studies with decreased transcription and translation of the CCND2 gene, we tested whether this opposite correlation exists in human cancers. Data from the Cancer Genome Atlas (TCGA) database indicated statistically significant negative correlation between PICOT and CCND2 in eight different human tumors where the highest correlation was in lung (p = 8.67E−10) and pancreatic (p = 1.06E−5) adenocarcinoma. Furthermore, high expression of PICOT and low expression of CCND2 correlated with poor patient survival in five different types of human tumors. The results suggest that PICOT binding to chromatin-associated EED modulates the H3K27me3 level at the CCND2 gene promoter which may be one of the potential mechanisms for regulation of cyclin D2 expression in tumors. These findings also indicate that a low PICOT/CCND2 expression ratio might serve as a good predictor of patient survival in selected human cancers.

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Survival Motor Neuron Protein Participates in Mouse Germ Cell Development and Spermatogonium Maintenance

International Journal of Molecular Sciences ◽

10.3390/ijms21030794 ◽

2020 ◽

Vol 21 (3) ◽

pp. 794 ◽

Cited By ~ 1

Author(s):

Wei-Fang Chang ◽

Jie Xu ◽

Tzu-Ying Lin ◽

Jing Hsu ◽

Hsiu-Mei Hsieh-Li ◽

...

Keyword(s):

Stem Cells ◽

Germ Cell ◽

Motor Neuron ◽

Cell Biology ◽

Critical Role ◽

Embryonic Stem ◽

Cell Development ◽

Survival Motor Neuron ◽

Specific Marker ◽

Germ Cell Development

The defective human survival motor neuron 1 (SMN1) gene leads to spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. We previously reported that loss of SMN results in rapid differentiation of Drosophila germline stem cells and mouse embryonic stem cells (ESCs), indicating that SMN also plays important roles in germ cell development and stem cell biology. Here, we show that in healthy mice, SMN is highly expressed in the gonadal tissues, prepubertal spermatogonia, and adult spermatocytes, whereas low SMN expression is found in differentiated spermatid and sperm. In SMA-like mice, the growth of testis tissues is retarded, accompanied with gamete development abnormalities and loss of the spermatogonia-specific marker. Consistently, knockdown of Smn1 in spermatogonial stem cells (SSCs) leads to a compromised regeneration capacity in vitro and in vivo in transplantation experiments. In SMA-like mice, apoptosis and accumulation of the R-loop structure were significantly elevated, indicating that SMN plays a critical role in the survival of male germ cells. The present work demonstrates that SMN, in addition to its critical roles in neuronal development, participates in mouse germ cell and spermatogonium maintenance.

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The Polycomb group protein CBX6 is an essential regulator of embryonic stem cell identity

Nature Communications ◽

10.1038/s41467-017-01464-w ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 16

Author(s):

Alexandra Santanach ◽

Enrique Blanco ◽

Hua Jiang ◽

Kelly R. Molloy ◽

Miriam Sansó ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Embryonic Stem ◽

Polycomb Group ◽

Polycomb Group Protein ◽

Cell Identity ◽

Group Protein

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SIRT1 affects DNA methylation of polycomb group protein target genes, a hotspot of the epigenetic shift observed in ageing

Human Genomics ◽

10.1186/s40246-015-0036-0 ◽

2015 ◽

Vol 9 (1) ◽

Cited By ~ 9

Author(s):

Luisa A Wakeling ◽

Laura J Ions ◽

Suzanne M Escolme ◽

Simon J Cockell ◽

Tianhong Su ◽

...

Keyword(s):

Dna Methylation ◽

Target Genes ◽

Polycomb Group ◽

Polycomb Group Protein ◽

Protein Target ◽

Group Protein

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The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.01432-06 ◽

2007 ◽

Vol 27 (10) ◽

pp. 3769-3779 ◽

Cited By ~ 461

Author(s):

Diego Pasini ◽

Adrian P. Bracken ◽

Jacob B. Hansen ◽

Manuela Capillo ◽

Kristian Helin

Keyword(s):

Cell Differentiation ◽

Transcriptional Activation ◽

Es Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Polycomb Group ◽

Embryonic Stem Cell Differentiation ◽

Es Cell ◽

Polycomb Group Protein ◽

Specific Expression

ABSTRACT Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12 −/− mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12 −/− ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12 −/− ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

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