Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation

Cegli, Rossella De; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; Bernardo, Diego di

doi:10.1093/nar/gks1136

Cited by 24 publications

(41 citation statements)

References 80 publications

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“…We found 1148 genes whose expression levels were altered by 2-fold or more in KO cells expressing WT EPOP vs. KO cells. These genes are associated with transcription, development and axon guidance (Table S1), similar to what has been described before (De Cegli et al, 2013). Gene set enrichment analysis (GSEA) revealed that PRC2 targets were significantly de-repressed independently of the interaction with Elongin BC (Figure 6B), consistent with our finding that Elongin BC is not required for EPOP to inhibit PRC2, and explains the partial but significant overlap of affected genes in WT versus the mutant EPOP-expressing cells (Figure 6C).…”

Section: Resultssupporting

confidence: 75%

“…C17orf96, esPRC2p48 and E130012A19Rik) (GeneID: 103551 (mouse); 100170841 (human))(UniprotID: Q7TNS8 (mouse); A6NHQ4 (human)) has recently been identified as an interacting partner of the mammalian PRC2 (Alekseyenko et al, 2014; De Cegli et al, 2013; Smits et al, 2013; Zhang et al, 2011) but its role and mechanism of action are poorly understood. It is highly expressed in ES cells, during embryogenesis and in the adult brain (Liefke and Shi, 2015) and has been proposed to contribute to somatic cell reprogramming (Zhang et al, 2011) and neuronal differentiation (De Cegli et al, 2013). We have found that EPOP represses PRC2 function in vivo, possibly by interfering with PRC2 chromatin binding via the interaction of its C-terminal region with the VEFS box of SUZ12 (Liefke and Shi, 2015).…”

Section: Introductionmentioning

confidence: 99%

“…EPOP also occupies actively transcribed genes and we observed a genome-wide alteration of H3K4me3 distribution upon EPOP knockdown, suggesting a global regulatory role (Liefke and Shi, 2015). Indeed, additional published work proposed EPOP as a pluripotency factor, which acts as a central regulatory hub in mESCs (De Cegli et al, 2013; Klein et al, 2015), implicating a more general regulatory role, beyond its function at PRC2 target genes. However, due to its unstructured and unique nature its precise role remains largely unknown (Liefke and Shi, 2015).…”

Section: Introductionmentioning

confidence: 99%

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EPOP Interacts with Elongin BC and USP7 to Modulate the Chromatin Landscape

2016

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Summary Gene regulatory networks are pivotal for many biological processes. In mouse embryonic stem cells (mESCs) the transcriptional network can be divided into three functionally distinct modules: Polycomb, Core and Myc. The Polycomb module represses developmental genes, while the Myc module is associated with proliferative functions and its mis-regulation is linked to cancer development. Here, we show that in mESCs the Polycomb Repressive Complex 2 (PRC2) associated protein EPOP (Elongin BC and Polycomb Repressive Complex 2-associated protein; a.k.a. C17orf96, esPRC2p48 and E130012A19Rik) co-localizes at chromatin with members of the Myc and Polycomb module. EPOP interacts with the transcription elongation factor Elongin BC and the H2B deubiquitinase USP7 to modulate transcriptional processes in mESCs similar to MYC. EPOP is commonly up-regulated in human cancer and its loss impairs the proliferation of several human cancer cell lines. Our findings establish EPOP as a transcriptional modulator, which impacts both Polycomb and active gene transcription in mammalian cells.

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Section: Resultssupporting

confidence: 75%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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EPOP Interacts with Elongin BC and USP7 to Modulate the Chromatin Landscape

2016

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“…We used 342 embryonic stem cell-specific genes that are both in our study and in the list of 543 genes ( F D R <.025) in [16]. There are 161 and 181 genes in each state.…”

Section: Resultsmentioning

confidence: 99%

Regression hidden Markov modeling reveals heterogeneous gene expression regulation: a case study in mouse embryonic stem cells

Lee

Ghosh

2014

BMC Genomics

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BackgroundStudies have shown the strong association between histone modification levels and gene expression levels. The detailed relationships between the two can vary substantially due to differential regulation, and hence a simple regression model may not be adequate. We apply a regression hidden Markov model (regHMM) to further investigate the potential multiple relationships between genes and histone methylation levels in mouse embryonic stem cells.ResultsSeven histone methylation levels are used in the study. Averaged histone modifications over non-overlapping 200 bp windows on the range transcription starting site (TSS) ± 1 Kb are used as predictors, and in total 70 explanatory variables are generated. Based on regHMM results, genes segregated into two groups, referred to as State 1 and State 2, have distinct association strengths. Genes in State 1 are better explained by histone methylation levels with R2=.72 while those in State 2 have weaker association strength with R2=.38. The regression coefficients in the two states are not very different in magnitude except in the intercept,.25 and 1.15 for State 1 and State 2, respectively. We found specific GO categories that may be attributed to the different relationships. The GO categories more frequently observed in State 2 match those of housekeeping genes, such as cytoplasm, nucleus, and protein binding. In addition, the housekeeping gene expression levels are significantly less explained by histone methylation in mouse embryonic stem cells, which is consistent with the constitutive expression patterns that would be expected.ConclusionGene expression levels are not universally affected by histone methylation levels, and the relationships between the two differ by the gene functions. The expression levels of the genes that perform the most common housekeeping genes’ GO categories are less strongly associated with histone methylation levels. We suspect that additional biological factors may also be strongly associated with the gene expression levels in State 2. We discover that the effect of the presence of CpG island in TSS ± 1 Kb is larger in State 2.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-360) contains supplementary material, which is available to authorized users.

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“…Most importantly, among the enriched EZH2 interactions we found two relatively uncharacterized human proteins: C10orf12, reported to be ubiquitously expressed (http://biogps.org/gene/26148/), and C17orf96, a potential homolog of mouse E13 (E130012A19Rik) protein implicated in epigenetic regulation of neuronal differentiation (15). C10orf12 and C17orf96 lack apparent homologs in Drosophila, but are conserved from bony fishes to humans, without discernible protein domains suggestive of their possible functions in the complex.…”

Section: Significancementioning

confidence: 99%

Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification

Alekseyenko

Gorchakov

Kharchenko

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Understanding the composition of epigenetic regulators remains an important challenge in chromatin biology. Traditional biochemical analysis of chromatin-associated complexes requires their release from DNA under conditions that can also disrupt key interactions. Here we develop a complementary approach (BioTAP-XL), in which cross-linking (XL) enhances the preservation of protein interactions and also allows the analysis of DNA targets under the same tandem affinity purification (BioTAP) regimen. We demonstrate the power of BioTAP-XL through analysis of human EZH2, a core subunit of polycomb repressive complex 2 (PRC2). We identify and validate two strong interactors, C10orf12 and C17orf96, which display enrichment with EZH2-BioTAP at levels similar to canonical PRC2 components (SUZ12, EED, MTF2, JARID2, PHF1, and AEBP2). ChIP-seq analysis of BioTAP-tagged C10orf12 or C17orf96 revealed the similarity of each binding pattern with the location of EZH2 and the H3K27me3-silencing mark, validating their physical interaction with PRC2 components. Interestingly, analysis by mass spectrometry of C10orf12 and C17orf96 interactions revealed that these proteins may be mutually exclusive PRC2 subunits that fail to interact with each other or with JARID2 and AEBP2. C10orf12, in addition, shows a strong and unexpected association with components of the EHMT1/2 complex, thus potentially connecting PRC2 to another histone methyltransferase. Similarly, results from CBX4-BioTAP protein pulldowns are consistent with reports of a diversity of PRC1 complexes. Our results highlight the importance of reciprocal analyses of multiple subunits and suggest that iterative use of BioTAP-XL has strong potential to reveal networks of chromatin-based interactions in higher organisms.he organization of the genome into active and silent domains is integral to the fidelity of gene regulation in higher organisms. Since their discovery in Drosophila, the genetic factors known collectively as the Polycomb Group (PcG) (1) have emerged as the prototypical epigenetic factors, required for the critical maintenance of gene silencing during development in higher organisms. Polycomb group proteins are known to form large multicomponent complexes that vary in their composition, with a broadly conserved distinction between PRC1 and PRC2 complexes (reviewed in ref.2). How these classes of key epigenetic factors are targeted to their sites of action, and interact with appropriate partners within their chromatin context, remains an important question.Biochemical analyses of PRC1 and PRC2 have been invaluable for the discoveries of enzymatic activities, specific binding properties, and strong subunit interactions (reviewed in refs. 2, 3). Recent seminal work on PRC1 has led to an emerging recognition of the diversity of PRC1 subcomplexes in mammals and their possible individual roles in transcriptional repression (4). These discoveries have highlighted the importance of dissecting chromatin-based complexes using multiple strategies. For example, it would be idea...

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Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation

Cited by 24 publications

References 80 publications

EPOP Interacts with Elongin BC and USP7 to Modulate the Chromatin Landscape

EPOP Interacts with Elongin BC and USP7 to Modulate the Chromatin Landscape

Regression hidden Markov modeling reveals heterogeneous gene expression regulation: a case study in mouse embryonic stem cells

Reciprocal interactions of human C10orf12 and C17orf96 with PRC2 revealed by BioTAP-XL cross-linking and affinity purification

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