PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus

AbstractPolycomb group (PcG) proteins maintain cell identity by repressing gene expression during development. Surprisingly, emerging studies have recently reported that a number of PcG proteins directly activate gene expression during cell fate determination process. However, the mechanisms by which they direct gene activation in pluripotency remain poorly understood. Here, we show that Phc1, a subunit of canonical polycomb repressive complex 1 (cPRC1), can exert its function in pluripotency maintenance via a PRC1-independent activation of Nanog. Ablation of Phc1 reduces the expression of Nanog and overexpression of Nanog partially rescues impaired pluripotency caused by Phc1 depletion. We find that Phc1 interacts with Nanog and activates Nanog transcription by stabilizing the genome-wide chromatin interactions of the Nanog locus. This adds to the already known canonical function of PRC1 in pluripotency maintenance via a PRC1-dependent repression of differentiation genes. Overall, our study reveals a function of Phc1 to activate Nanog transcription through regulating chromatin architecture and proposes a paradigm for PcG proteins to maintain pluripotency.

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Analysis of Polycomb Repressive Complexes binding dynamics during limb development reveals the prevalence of PRC2-independent PRC1 occupancy

10.1101/2020.09.21.306688 ◽

2020 ◽

Author(s):

Claudia Gentile ◽

Alexandre Mayran ◽

Fanny Guerard-Millet ◽

Marie Kmita

Keyword(s):

Gene Expression ◽

Limb Development ◽

Functional Relationship ◽

Specific Activity ◽

Polycomb Group ◽

Specific Gene ◽

Developmental Genes ◽

Genome Wide ◽

Polycomb Repressive Complexes ◽

Polycomb Repressive Complex 1

AbstractThe Polycomb group (PcG) proteins are key players in the regulation of tissue-specific gene expression through their known ability to epigenetically silence developmental genes. The PcG proteins form two multicomponent complexes, Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2), whereby the hierarchical model of recruitment postulates that PRC2 triggers the trimethylation of Histone H3 lysine 27 (H3K27me3) leading to the recruitment of PRC1. Here we report on the genome-wide binding dynamics of components from both PRC1 and PRC2 in the developing limb. We show that a large proportion of PRC-bound promoters are occupied exclusively by PRC1, suggesting a more extensive PRC1-specific activity than anticipated. We found that PRC1 (RING1B) and PRC2 (SUZ12) co-occupy the promoters of developmental genes, for which a subset become up-regulated upon the inactivation of PRC2. Strikingly, we found that RING1B occupancy is largely unaffected by the loss of PRC2, revealing a complex functional relationship between these two complexes in regulating gene expression and possibly an expansive functional interplay between canonical and non-canonical PRC1.

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Functional characterization of human Polycomb-like 3 isoforms identifies them as components of distinct EZH2 protein complexes

Biochemical Journal ◽

10.1042/bj20100944 ◽

2011 ◽

Vol 434 (2) ◽

pp. 333-342 ◽

Cited By ~ 26

Author(s):

Gaylor Boulay ◽

Claire Rosnoblet ◽

Cateline Guérardel ◽

Pierre-Olivier Angrand ◽

Dominique Leprince

Keyword(s):

Cell Fate ◽

Target Genes ◽

Protein Complexes ◽

Functional Characterization ◽

Polycomb Group ◽

Phd Finger ◽

Reverse Transcription Pcr ◽

Tudor Domain ◽

Self Association ◽

Polycomb Repressive Complex 1

PcG (Polycomb group) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2–4. In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (Polycomb-like). In the present study, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L, containing an N-terminal TUDOR domain and two PHDs (plant homeodomains) and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative reverse transcription–PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medulloblastoma. By co-immunoprecipitation analyses, we demonstrated that both isoforms interact with EZH2 through their common TUDOR domain. However, the hPCL3L-specific PHD2 domain, which is better conserved than PHD1 in the PCL family, is also involved in this interaction and implicated in the self-association of hPCL3L. Finally, we have demonstrated that both hPCL3 isoforms are physically associated with EZH2, but in different complexes. Our results provide the first evidence that the two hPCL3 isoforms belong to different complexes and raise important questions about their relative functions, particularly in tumorigenesis.

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H4K20me3 methyltransferase SUV420H2 shapes the chromatin landscape of pluripotent embryonic stem cells

Development ◽

10.1242/dev.188516 ◽

2020 ◽

Vol 147 (23) ◽

pp. dev188516

Author(s):

Jiji T. Kurup ◽

Zhijun Han ◽

Wenfei Jin ◽

Benjamin L. Kidder

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Es Cells ◽

Three Dimensional ◽

Embryonic Stem ◽

Pericentric Heterochromatin ◽

Es Cell ◽

Chromatin Interactions ◽

Repressive Histone Modification ◽

Dna Elements

ABSTRACTHeterochromatin, a densely packed chromatin state that is transcriptionally silent, is a critical regulator of gene expression. However, it is unclear how the repressive histone modification H4K20me3 or the histone methyltransferase SUV420H2 regulates embryonic stem (ES) cell fate by patterning the epigenetic landscape. Here, we report that depletion of SUV420H2 leads to a near-complete loss of H4K20me3 genome wide, dysregulated gene expression and delayed ES cell differentiation. SUV420H2-bound regions are enriched with repetitive DNA elements, which are de-repressed in SUV420H2 knockout ES cells. Moreover, SUV420H2 regulation of H4K20me3-marked heterochromatin controls chromatin architecture, including fine-scale chromatin interactions in pluripotent ES cells. Our results indicate that SUV420H2 plays a crucial role in stabilizing the three-dimensional chromatin landscape of ES cells, as loss of SUV420H2 resulted in A/B compartment switching, perturbed chromatin insulation, and altered chromatin interactions of pericentric heterochromatin and surrounding regions, indicative of localized decondensation. In addition, depletion of SUV420H2 resulted in compromised interactions between H4K20me3 and gene-regulatory regions. Together, these findings describe a new role for SUV420H2 in regulating the chromatin landscape of ES cells.

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The transcription factor Schnurri plays a dual role in mediating Dpp signaling during embryogenesis

Development ◽

10.1242/dev.128.9.1657 ◽

2001 ◽

Vol 128 (9) ◽

pp. 1657-1670 ◽

Cited By ~ 1

Author(s):

J. Torres-Vazquez ◽

S. Park ◽

R. Warrior ◽

K. Arora

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Cell Fate ◽

Target Genes ◽

Gene Activation ◽

Nuclear Accumulation ◽

Embryonic Patterning ◽

Specific Analysis ◽

Positive Input ◽

Affect Gene Expression

Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenic protein 2/4, is crucial for embryonic patterning and cell fate specification in Drosophila. Dpp signaling triggers nuclear accumulation of the Smads Mad and Medea, which affect gene expression through two distinct mechanisms: direct activation of target genes and relief of repression by the nuclear protein Brinker (Brk). The zinc-finger transcription factor Schnurri (Shn) has been implicated as a co-factor for Mad, based on its DNA-binding ability and evidence of signaling dependent interactions between the two proteins. A key question is whether Shn contributes to both repression of brk as well as to activation of target genes. We find that during embryogenesis, brk expression is derepressed in shn mutants. However, while Mad is essential for Dpp-mediated repression of brk, the requirement for shn is stage specific. Analysis of brk; shn double mutants reveals that upregulation of brk does not account for all aspects of the shn mutant phenotype. Several Dpp target genes are expressed at intermediate levels in double mutant embryos, demonstrating that shn also provides a brk-independent positive input to gene activation. We find that Shn-mediated relief of brk repression establishes broad domains of gene activation, while the brk-independent input from Shn is crucial for defining the precise limits and levels of Dpp target gene expression in the embryo.

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Single Cell Level Genome-Wide Gene Expression Analysis of Bone Marrow-Derived Oct-4+ very Small Embryonic-Like Stem Cells (VSELs) Revealed That a Polycomb Group Protein Ezh2 Regulates VSELs Pluripotency by Maintaining Bivalent Domains At Promoters of Important Homeodomain-Containing Developmental Transcription Factors

Blood ◽

10.1182/blood.v118.21.2345.2345 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2345-2345

Author(s):

Magda Kucia ◽

Rui Liu ◽

Kasia Mierzejewska ◽

Wan Wu ◽

Janina Ratajczak ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Transcription Factors ◽

Single Cell ◽

Gene Expression Analysis ◽

Polycomb Group ◽

Polycomb Group Protein ◽

Genome Wide ◽

Group Protein ◽

Genome Wide Gene Expression

Abstract Abstract 2345 Recently, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult bone marrow (BM) (Leukemia 2006:20;857). These Oct4+CXCR4+SSEA-1+Sca-1+CD45−Lin− VSELs are capable of differentiation in vitro into cells from all three germ lineages and in in vivo animal models they can be specified into mesenchymal stem cells (MSCs) (Stem Cells Dev 2010:19;1557), cardiomyocytes (Stem Cell 2008:26;1646), and long-term engrafting hematopoietic stem cells (HSCs) (Exp Hematol 2011:39;225). Be employing gene-expression and epigenetic profiling studies we reported that VSELs in BM have germ-line stem cell like epigenetic features including i) open/active chromatin structure in Oct4 promoter, ii) parent-of-origin specific reprogramming of genomic imprinting (Leukemia 2009, 23, 2042–2051), and iii) that they share several markers with epiblast-derived primordial germ cells (PGCs), in particular with migratory PGCs (Leukemia 2010, 24, 1450–1461). However, it was not clear how VSELs maintain pluripotent state. To address this issue we recently employed single cell-based genome-wide gene expression analysis and found that, Oct4+ VSELs i) express a similar, yet nonidentical, transcriptome as embryonic stem-cells (ESCs), ii) up-regulate cell-cycle checkpoint genes, and iii) down-regulate genes involved in protein turnover and mitogenic pathways. Interestingly, our single cell library studies also revelaed that Ezh2, a polycomb group protein, is highly expressed in VSELs. This protein is well known to be involved in maintaining a bivalent domains (BDs) at promoters of important homeodomain-containing developmental transcription factors. Of note a presence of BDs is characteristic for pluripotent stem cells (e.g., ESCs) and as result of Ezh2 overexpression, VSELs, like ESCs, exhibit BDs - bivalently modified nucleosomes (trimethylated H3K27 and H3K4) at promoters of important homeodomain-containing developmental transcription factors (Sox21 Nkx2.2 Dlx1 Zfpm2 Irx2 Lbx1h Hlxb9 Pax5 HoxA3). Of note, spontaneous (as seen during differentiation) or RNA interference-enforced down-regulation of Ezh2 removes BDs what, results in lose of their plurioptentiality and de-repression of several BD-regulated genes that control their tissue commitment. In conclusion, Our results show for first time that in addition to the expression of pluripotency core transcription factor Oct-4, VSELs, like other pluripotent stem-cells, maintain their pluripotent state through an Ezh2-dependent BD-mediated epigenetic mechanism. Based on this our genome-wide gene expression study not only advances our understanding of biological processes that govern VSELs pluripotency, differentiation, and quiescence but will also help to develop better protocols for ex vivo expansion of these promising cells for potential application in regenerative medicine. Disclosures: Ratajczak: Neostem Inc: Consultancy, Research Funding.

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Polycomb-Mediated Chromatin Loops Revealed by a Sub-Kilobase Resolution Chromatin Interaction Map

10.1101/099804 ◽

2017 ◽

Cited By ~ 5

Author(s):

Kyle P. Eagen ◽

Erez Lieberman Aiden ◽

Roger D. Kornberg

Keyword(s):

Polycomb Group ◽

Dna Looping ◽

Gene Repression ◽

Chromatin Interaction ◽

Chromatin Loops ◽

A Subunit ◽

High Throughput Dna Sequencing ◽

Interaction Map ◽

Polycomb Repressive Complex 1 ◽

Chemical Cross Linking

ABSTRACTThe locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or “anchors” are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of Polycomb Repressive Complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer-promoter communication, our results indicate that loops are also associated with gene repression.

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Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence

10.1101/2020.04.28.065888 ◽

2020 ◽

Author(s):

Lisa Muniz ◽

Sandra Lazorthes ◽

Maxime Delmas ◽

Julien Ouvrard ◽

Marion Aguirrebengoa ◽

...

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Protein Localization ◽

Polycomb Group ◽

Circular Rnas ◽

Polycomb Proteins ◽

Cycle Arrest ◽

Polycomb Protein ◽

Stable Species ◽

Non Coding Rnas

AbstractLong non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence.

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Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes

eLife ◽

10.7554/elife.02637 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 32

Author(s):

Roberto Bonasio ◽

Emilio Lecona ◽

Varun Narendra ◽

Philipp Voigt ◽

Fabio Parisi ◽

...

Keyword(s):

Gene Expression ◽

Epigenetic Regulation ◽

Target Genes ◽

Rna Binding ◽

Regulation Of Gene Expression ◽

Polycomb Group ◽

Polycomb Group Protein ◽

Binding Region ◽

Group Protein ◽

Polycomb Repressive Complex 1

Polycomb repressive complex-1 (PRC1) is essential for the epigenetic regulation of gene expression. SCML2 is a mammalian homolog of Drosophila SCM, a Polycomb-group protein that associates with PRC1. In this study, we show that SCML2A, an SCML2 isoform tightly associated to chromatin, contributes to PRC1 localization and also directly enforces repression of certain Polycomb target genes. SCML2A binds to PRC1 via its SPM domain and interacts with ncRNAs through a novel RNA-binding region (RBR). Targeting of SCML2A to chromatin involves the coordinated action of the MBT domains, RNA binding, and interaction with PRC1 through the SPM domain. Deletion of the RBR reduces the occupancy of SCML2A at target genes and overexpression of a mutant SCML2A lacking the RBR causes defects in PRC1 recruitment. These observations point to a role for ncRNAs in regulating SCML2 function and suggest that SCML2 participates in the epigenetic control of transcription directly and in cooperation with PRC1.

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No kissing in the nucleus: Unbiased analysis reveals no evidence of trans chromosomal regulation of mammalian immune development

10.1101/212985 ◽

2017 ◽

Cited By ~ 3

Author(s):

Timothy M. Johanson ◽

Hannah D. Coughlan ◽

Aaron T.L. Lun ◽

Naiara G. Bediaga ◽

Gaetano Naselli ◽

...

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Immune Cells ◽

Chromosome Conformation Capture ◽

Chromosome Conformation ◽

Immune Development ◽

Regulate Gene Expression ◽

Genome Wide ◽

Capture Technique ◽

Regulate Gene

SummaryIt has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions.

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Gene Regulatory Networks Governing Hematopoietic Stem Cell Development and Identity

Blood ◽

10.1182/blood.v118.21.sci-30.sci-30 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-30-SCI-30 ◽

Cited By ~ 1

Author(s):

Tariq Enver

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Single Cells ◽

Transcriptional Regulators ◽

Gata Factor ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Genome Wide ◽

A Genome ◽

Custom Made

Abstract Abstract SCI-30 Several studies have addressed questions about transcriptional regulation within particular hematopoietic cell compartments. Few, however, have attempted to capture the transcriptional changes that occur during the dynamic transition from one compartment to another. We have profiled gene expression as multipotential progenitors underwent commitment and differentiation to two alternative lineages, focusing on the first 3 days of differentiation when the majority of decisions about cell fate are made. We have combined this with genome-wide identification of the targets of three key transcription factors before and after differentiation; GATA-2, usually associated with the stem/progenitor compartment; GATA-1 (erythroid); and PU.1 (myeloid). These data have been compiled into a custom-made queryable database, designed to be intuitive to use and to provide tools to interrogate the data on many levels. We used correlation analyses to associate transcription factor binding with particular modules of co-expressed genes, alongside detailed sequence analysis of bound regions. These approaches have informed our understanding of GATA factor switching, and highlighted novel roles for both GATA-2 and Pu.1 in erythroid cells. Overall, the data reveal greater degree of complexity in the interplay between these three factors in regulating hematopoiesis than has hitherto been described, and highlights the importance of a genome-wide approach to understanding complex regulatory systems. A significant challenge in the field is how to relate these types of population-based data to the action of transcriptional regulators within single cells where cell fate decisions ultimately are affected. As a step toward this, we have generated single cell profiles of gene expression for a limited set of transcriptional regulators in self-renewing and committed blood cells and used these data to build a stochastic computational model, which affords exploration of commitment scenarios in silico. Disclosures: No relevant conflicts of interest to declare.

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