Epigenetic Regulation of Intestinal Stem Cells and Disease: A Balancing Act of DNA and Histone Methylation

Genetic mutations or regulatory failures underlie cellular malfunction in many diseases, including colorectal cancer and inflammatory bowel diseases. However, mutational defects alone fail to explain the complexity of such disorders. Epigenetic regulation—control of gene action through chemical and structural changes of chromatin—provides a platform to integrate multiple extracellular inputs and prepares the cellular genome for appropriate gene expression responses. Coregulation by polycomb repressive complex 2–mediated trimethylation of lysine 27 on histone 3 and DNA methylation has emerged as one of the most influential epigenetic controls in colorectal cancer and many other diseases, but molecular details remain inadequate. Here we review the molecular interplay of these epigenetic features in relation to gastrointestinal development, homeostasis, and disease biology. We discuss other epigenetic mechanisms pertinent to the balance of trimethylation of lysine 27 on histone 3 and DNA methylation and their actions in gastrointestinal cancers. We also review the current molecular understanding of chromatin control in the pathogenesis of inflammatory bowel diseases. Genetic mutations or regulatory failures underlie cellular malfunction in many diseases, including colorectal cancer and inflammatory bowel diseases. However, mutational defects alone fail to explain the complexity of such disorders. Epigenetic regulation—control of gene action through chemical and structural changes of chromatin—provides a platform to integrate multiple extracellular inputs and prepares the cellular genome for appropriate gene expression responses. Coregulation by polycomb repressive complex 2–mediated trimethylation of lysine 27 on histone 3 and DNA methylation has emerged as one of the most influential epigenetic controls in colorectal cancer and many other diseases, but molecular details remain inadequate. Here we review the molecular interplay of these epigenetic features in relation to gastrointestinal development, homeostasis, and disease biology. We discuss other epigenetic mechanisms pertinent to the balance of trimethylation of lysine 27 on histone 3 and DNA methylation and their actions in gastrointestinal cancers. We also review the current molecular understanding of chromatin control in the pathogenesis of inflammatory bowel diseases. Gastrointestinal stem cells produce billions of epithelial cells of discrete types each day. In intestine and colon, these cells continuously make precise cell fate decisions through hundreds of well-orchestrated gene expression changes to maintain tissue homeostasis and in response to injuries. While doing so, the cells receive various extracellular stimuli from the chemical and microbial milieu of the lumen, and from the underlying mesenchyme. Cell response to the external environment is significantly influenced by intrinsic cellular variables, including the genome and the epigenome. Although the genome is largely stable, the epigenome allows for dynamic control of gene expression and cellular identity. Accordingly, varying levels of genetic as well as epigenetic changes contribute to gastrointestinal diseases, such as colorectal cancer (CRC) and inflammatory bowel disease (IBD). Methylation of DNA and histone modifications, such as methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation, are incorporated by various enzymes as post-translational modifications. Depending on their co-occupancy and placement along the genome, these modifications influence the surrounding chromatin structure, access to transcription factors (TFs) and enzymes, and gene expression. Both DNA methylation and polycomb repressive complex 2 (PRC2)–mediated histone 3 lysine 27 trimethylation (H3K27me3) are important epigenetic modifications associated with cell-type–specific gene repression during tissue development and homeostasis. In addition, their functional interaction with each other and many other chromatin features has been identified as a major contributing factor to CRC initiation and progression.1Baylin S.B. Jones P.A. Epigenetic determinants of cancer.Cold Spring Harb Perspect Biol. 2016; 8a019505Crossref PubMed Scopus (177) Google Scholar Here we focus on how their combined action influences intestinal function and diseases. Recent studies have provided unprecedented detail regarding PRC2 composition and its effect on chromatin modification and function. PRC2 is a multimeric protein complex that consists of core subunits EZH1/2, EED, SUZ12, and RBBP4/7. The catalytic domain of PRC2 is encoded by SET-domain proteins EZH1 or EZH2.2Montgomery N.D. Yee D. Chen A. et al.The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation.Curr Biol. 2005; 15: 942-947Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 3Healy E. Mucha M. Glancy E. et al.PRC2.1 and PRC2.2 synergize to coordinate H3K27 trimethylation.Mol Cell. 2019; 76: 437-452.e6Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 4Cao R. Zhang Y. SUZ12 is required for both the histone methyltransferase activity and the silencing function of the EED-EZH2 complex.Mol Cell. 2004; 15: 57-67Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar, 5Kasinath V. Faini M. Poepsel S. et al.Structures of human PRC2 with its cofactors AEBP2 and JARID2.Science. 2018; 359: 940-944Crossref PubMed Scopus (105) Google Scholar However, the enzymatic activity of PRC2 is also dependent on EED, SUZ12, and histone-binding proteins, making PRC2 an obligate quadromeric complex. PRC2 can mono-, di-, or tri-methylate lysine 27 of histone H3 and is the only identified H3K27 methyltransferase. PRC2-mediated H3K27me3 deposition at gene promoters and gene bodies forms repressive chromatin state. Thus, appropriate distribution of H3K27me3 across the genome is essential for cell-type–specific gene expression and formation of cell identity during differentiation in embryonic stem cells (ESCs) and adult stem cells.6Margueron R. Reinberg D. The polycomb complex PRC2 and its mark in life.Nature. 2011; 469: 343-349Crossref PubMed Scopus (2116) Google Scholar,7Yang X. Hu B. Hou Y. et al.Silencing of developmental genes by H3K27me3 and DNA methylation reflects the discrepant plasticity of embryonic and extraembryonic lineages.Cell Res. 2018; 28: 593-596Crossref PubMed Scopus (17) Google Scholar There are 2 recognized subtypes of PRC2, PRC2.1 and PRC2.2, they differ in composition of their accessory proteins.3Healy E. Mucha M. Glancy E. et al.PRC2.1 and PRC2.2 synergize to coordinate H3K27 trimethylation.Mol Cell. 2019; 76: 437-452.e6Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,8Holoch D. Margueron R. Mechanisms regulating PRC2 recruitment and enzymatic activity.Trends Biochem Sci. 2017; 42: 531-542Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar PRC2.1 is associated with PCL1/PHF1, PCL2/MTF2, or PCL3/PHF19 and EPOP or PALI1. However, PRC2.2 contains JARID2 and AEBP2 as accessory proteins. PRC2.1 and PRC2.2 synergy is critical for proper disposition of H3K27me3 and inhibition of their associated proteins cause redistribution of H3K27me3.3Healy E. Mucha M. Glancy E. et al.PRC2.1 and PRC2.2 synergize to coordinate H3K27 trimethylation.Mol Cell. 2019; 76: 437-452.e6Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Although, the majority of the PRC2.1 and PRC2.2 target sites overlap, they direct H3K27me3 deposition in an independent manner. PRC2.2 chromatin localization is dependent on PRC1 activity and mediated through binding of JARID2 to H2AK119Ub.3Healy E. Mucha M. Glancy E. et al.PRC2.1 and PRC2.2 synergize to coordinate H3K27 trimethylation.Mol Cell. 2019; 76: 437-452.e6Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,9Cooper S. Grijzenhout A. Underwood E. et al.Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between polycomb complexes PRC1 and PRC2.Nat Commun. 2016; 7: 13661Crossref PubMed Scopus (138) Google Scholar The structure and mechanism of PRC2 is reviewed extensively elsewhere.10Laugesen A. Højfeldt J.W. Helin K. Molecular mechanisms directing PRC2 recruitment and H3K27 methylation.Mol Cell. 2019; 74: 8-18Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar DNA methylation represents one of the most studied and well-understood heritable epigenetic changes of the DNA. The de novo DNA methylases DNAMT3A and DNMT3B modify more than 28 million CpG bases across the human genome through covalent addition of a methyl group, and DNMT1 maintains the modification by faithfully copying these patterns over cell divisions. DNA methylation can be actively removed through oxidation by TET1, TET2, and TET3 enzymes (ten-eleven translocations).11Wu H. Zhang Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation.Genes Dev. 2011; 25: 2436-2452Crossref PubMed Scopus (497) Google Scholar Traditionally, studies have focused on understanding the effects of DNA methylation patterns at genes and their promoters: unmethylated promoters of active genes, methylation across gene bodies, and hypermethylation at promoters leading to gene repression in cancers. A significant addition to this regulatory view of DNA methylation was the observation that cis-regulatory regions or enhancers are hypomethylated.12Domcke S. Bardet A.F. Adrian Ginno P. et al.Competition between DNA methylation and transcription factors determines binding of NRF1.Nature. 2015; 528: 575-579Crossref PubMed Scopus (269) Google Scholar This local demethylation is induced by binding of DNA methylation–insensitive TFs, which further enables binding of other methylation-sensitive TFs. Thus, tissue-specific enhancer formation is linked with DNA methylation changes dependent on coregulatory TF action. Studies examining PRC2 targeting and H3K27me3 deposition in intestinal development, homeostasis, and intestinal stem cell differentiation reveal how PRC2 and DNA methylation co-govern tissue function.13Jadhav U. Cavazza A. Banerjee K.K. et al.Extensive recovery of embryonic enhancer and gene memory stored in hypomethylated enhancer DNA.Mol Cell. 2019; 74: 542-554.e5Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,14Jadhav U. Manieri E. Nalapareddy K. et al.Replicational dilution of H3K27me3 in mammalian cells and the role of poised promoters.Mol Cell. 2020; 78: 141-151.e5Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In addition, studies in ESCs and other cells have revealed novel compositions of PRC2 and significant mechanistic details regarding its function.10Laugesen A. Højfeldt J.W. Helin K. Molecular mechanisms directing PRC2 recruitment and H3K27 methylation.Mol Cell. 2019; 74: 8-18Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar We discuss the relevant studies and their implications for future exploration of epigenetic control in diseases, including CRC and IBD. The roles of DNA and H3K27 methylation have been tested in detail in the intestine. Loss of DNA methylation through disruption of methyltransferase Dnmt1 activity in mouse intestinal epithelium causes crypt expansion due to delayed differentiation, but intestinal function remains unperturbed.15Sheaffer K.L. Kim R. Aoki R. et al.DNA methylation is required for the control of stem cell differentiation in the small intestine.Genes Dev. 2014; 28: 652-664Crossref PubMed Scopus (134) Google Scholar Ezh2, the enzymatic subunit of PRC2, is expressed at the highest level in the crypt of the intestine, where all of the cycling cells, including the intestinal stem cells, reside.16Jadhav U. Nalapareddy K. Saxena M. et al.Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells.Cell. 2016; 165: 1389-1400Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Genomic deletion of Eed causes complete loss of PRC2 action and leads to cell cycle arrest in crypts, which can be attributed to the loss of H3K27me3-based repression at tumor suppressor Cdkn2a.14Jadhav U. Manieri E. Nalapareddy K. et al.Replicational dilution of H3K27me3 in mammalian cells and the role of poised promoters.Mol Cell. 2020; 78: 141-151.e5Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,16Jadhav U. Nalapareddy K. Saxena M. et al.Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells.Cell. 2016; 165: 1389-1400Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Koppens M.A. Bounova G. Gargiulo G. et al.Deletion of polycomb repressive complex 2 from mouse intestine causes loss of stem cells.Gastroenterology. 2016; 151: 684-697.e12Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Chiacchiera F. Rossi A. Jammula S. et al.PRC2 preserves intestinal progenitors and restricts secretory lineage commitment.EMBO J. 2016; 35: 2301-2314Crossref PubMed Scopus (48) Google Scholar Surprisingly, very few genes involved in intestinal stem cell differentiation and homeostasis are controlled by PRC2. Instead, genes expressed during the intestinal development are silenced by H3K27me3 in adult epithelium and get reactivated in the absence of PRC2 action.13Jadhav U. Cavazza A. Banerjee K.K. et al.Extensive recovery of embryonic enhancer and gene memory stored in hypomethylated enhancer DNA.Mol Cell. 2019; 74: 542-554.e5Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar,16Jadhav U. Nalapareddy K. Saxena M. et al.Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells.Cell. 2016; 165: 1389-1400Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar This reactivation is highly influenced by functional interactions of H3K27me3 with other histone modifications and DNA methylation. Activating histone modification H3K4me3 is found at many gene promoters decorated with H3K27me3, resulting in formation of bivalent domains. The amount of H3K4me3 mark at bivalent promoters of developmental genes can predict the extent of gene activation upon PRC2 loss.14Jadhav U. Manieri E. Nalapareddy K. et al.Replicational dilution of H3K27me3 in mammalian cells and the role of poised promoters.Mol Cell. 2020; 78: 141-151.e5Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,16Jadhav U. Nalapareddy K. Saxena M. et al.Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells.Cell. 2016; 165: 1389-1400Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Moreover, decommissioned enhancers of these developmental genes maintain DNA hypomethylation in adult cells, and get activated in PRC2 null cells.13Jadhav U. Cavazza A. Banerjee K.K. et al.Extensive recovery of embryonic enhancer and gene memory stored in hypomethylated enhancer DNA.Mol Cell. 2019; 74: 542-554.e5Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar These novel observations in the adult and developmental intestine reveal principles of PRC2 function in adult mammalian cells. However, many fundamental questions, including how mammalian PRC2 is recruited to its genomic target sites, are still a point of contention in the field of epigenomics. In Drosophila, the polycomb complex is recruited to specific genomic regions called the polycomb responsive elements, but no such regions are identified in mammalian genomes.19Kassis J.A. Brown J.L. Polycomb group response elements in Drosophila and vertebrates.Adv Genet. 2013; 81: 83-118Crossref PubMed Scopus (139) Google Scholar Sequence specificity is likely not a major factor in recruitment of PRC2 to the mammalian genome because no recurrent motif has been associated with PRC2 binding. This further emphasizes the degree of complexity regulating PRC2 recruitment and activity; the underlying molecular mechanism likely involve DNA methylation, nucleosomal localization, as well as the ability of H3K27me3 to colocalize with both permissive and repressive histone modifications like H3K4me3 and H3K9me3, respectively.20Bernstein B.E. Mikkelsen T.S. Xie X. et al.A bivalent chromatin structure marks key developmental genes in embryonic stem cells.Cell. 2006; 125: 315-326Abstract Full Text Full Text PDF PubMed Scopus (3941) Google Scholar, 21Lorzadeh A. Bilenky M. Hammond C. et al.Nucleosome density ChIP-seq identifies distinct chromatin modification signatures associated with MNase accessibility.Cell Rep. 2016; 17: 2112-2124Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 22Pellacani D. Bilenky M. Kannan N. et al.Analysis of normal human mammary epigenomes reveals cell-specific active enhancer states and associated transcription factor networks.Cell Rep. 2016; 17: 2060-2074Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar In addition, H3K27me3 can also act to restrict spreading of epigenetic modifications, such as the activating mark H3K36me2,23Lu C. Jain S.U. Hoelper D. et al.Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape.Science. 2016; 352: 844-849Crossref PubMed Google Scholar or form “bivalent” enhancers hosting both H3K27me3 and H3K4me1 at intergenic regions.14Jadhav U. Manieri E. Nalapareddy K. et al.Replicational dilution of H3K27me3 in mammalian cells and the role of poised promoters.Mol Cell. 2020; 78: 141-151.e5Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar,16Jadhav U. Nalapareddy K. Saxena M. et al.Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells.Cell. 2016; 165: 1389-1400Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar,20Bernstein B.E. Mikkelsen T.S. Xie X. et al.A bivalent chromatin structure marks key developmental genes in embryonic stem cells.Cell. 2006; 125: 315-326Abstract Full Text Full Text PDF PubMed Scopus (3941) Google Scholar,24Kundaje A. Meuleman W. Ernst J. et al.Integrative analysis of 111 reference human epigenomes.Nature. 2015; 518: 317-330Crossref PubMed Scopus (3337) Google Scholar Bivalent states likely poise expression of developmental genes in pluripotent and multipotent cells. Abnormal PRC2 activity can disrupt the balance of activation and deactivation of these unique chromatin states, affecting stem cell differentiation and proliferation and resulting in adverse outcomes, including tumor formation. It is not surprising that modulation of PRC2 and H3K27me3, through mutations or indirect chromatin deregulation, is a recurrent observation in many cancer types, including CRC. More than half of all mammalian genes contain high-density CpG regions (termed CpG islands [CGIs]) around their promoters. H3K27me3-mediated repression is a recurrent event at the hypomethylated CGI promoters (Figure 1A), which highlights the tug of war between DNA methylation and H3K27me3 for transcriptional repression.21Lorzadeh A. Bilenky M. Hammond C. et al.Nucleosome density ChIP-seq identifies distinct chromatin modification signatures associated with MNase accessibility.Cell Rep. 2016; 17: 2112-2124Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,25Jermann P. Hoerner L. Burger L. et al.Short sequences can efficiently recruit histone H3 lysine 27 trimethylation in the absence of enhancer activity and DNA methylation.Proc Natl Acad Sci U S A. 2014; 111: E3415-E3421Crossref PubMed Scopus (96) Google Scholar The dilemma of the competition between these 2 repressive epigenetic modifications for genomic real estate is an active field of research.21Lorzadeh A. Bilenky M. Hammond C. et al.Nucleosome density ChIP-seq identifies distinct chromatin modification signatures associated with MNase accessibility.Cell Rep. 2016; 17: 2112-2124Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,26McLaughlin K. Flyamer I.M. Thomson J.P. et al.DNA methylation directs polycomb-dependent 3D genome re-organization in naive pluripotency.Cell Rep. 2019; 29: 1974-1985.e6Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 27Inoue A. Jiang L. Lu F. et al.Maternal H3K27me3 controls DNA methylation-independent imprinting.Nature. 2017; 547: 419-424Crossref PubMed Scopus (199) Google Scholar, 28Boulard M. Edwards J.R. Bestor T.H. FBXL10 protects polycomb-bound genes from hypermethylation.Nat Genet. 2015; 47: 479-485Crossref PubMed Scopus (98) Google Scholar The mutual antagonism of H3K27me3 and DNA methylation at CGI promoters suggests that PRC2-targeted CGI promoters require protection from DNA methylation by chromosomal proteins, as in the case of Fbxl10 in mouse embryos28Boulard M. Edwards J.R. Bestor T.H. FBXL10 protects polycomb-bound genes from hypermethylation.Nat Genet. 2015; 47: 479-485Crossref PubMed Scopus (98) Google Scholar or other active histone modifications, as in the case of H3K4me3 at bivalent promoters21Lorzadeh A. Bilenky M. Hammond C. et al.Nucleosome density ChIP-seq identifies distinct chromatin modification signatures associated with MNase accessibility.Cell Rep. 2016; 17: 2112-2124Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,29Neri F. Krepelova A. Incarnato D. et al.Dnmt3L antagonizes DNA methylation at bivalent promoters and favors DNA methylation at gene bodies in ESCs.Cell. 2013; 155: 121-134Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar (Figure 1B). Perhaps the dual influence of H3K27me3 and DNA methylation at CGI promoters tune stem cell plasticity, where H3K27me3 and DNA methylation advocate plastic and rigid cellular identity, respectively. After all, H3K27me3 plays a critical role in maintaining bivalency at promoters, a major contributor to cell plasticity required for differentiation, in both ESCs20Bernstein B.E. Mikkelsen T.S. Xie X. et al.A bivalent chromatin structure marks key developmental genes in embryonic stem cells.Cell. 2006; 125: 315-326Abstract Full Text Full Text PDF PubMed Scopus (3941) Google Scholar and adult stem cells.16Jadhav U. Nalapareddy K. Saxena M. et al.Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells.Cell. 2016; 165: 1389-1400Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Moreover, expression of many of the developmental regulatory genes, including SOX2, NODAL, and EOMES, are regulated by H3K27me3 in ESCs, mesenchymal stem cells, and neurospheres.30Xie W. Schultz M.D. Lister R. et al.Epigenomic analysis of multilineage differentiation of human embryonic stem cells.Cell. 2013; 153: 1134-1148Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar Even in naïve pluripotent cells, H3K27me3 and DNA methylation regulate each other’s distribution and dictate higher-order chromatin structure.26McLaughlin K. Flyamer I.M. Thomson J.P. et al.DNA methylation directs polycomb-dependent 3D genome re-organization in naive pluripotency.Cell Rep. 2019; 29: 1974-1985.e6Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar,27Inoue A. Jiang L. Lu F. et al.Maternal H3K27me3 controls DNA methylation-independent imprinting.Nature. 2017; 547: 419-424Crossref PubMed Scopus (199) Google Scholar,31Brinkman A.B. Gu H. Bartels S.J. et al.Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk.Genome Res. 2012; 22: 1128-1138Crossref PubMed Scopus (270) Google Scholar DNA methylation also co-occurs with epigenetic layers that restrict spreading of repressive H3K27me3, such as H3K36 methylation23Lu C. Jain S.U. Hoelper D. et al.Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape.Science. 2016; 352: 844-849Crossref PubMed Google Scholar,32Weinberg D.N. Papillon-Cavanagh S. Chen H. et al.The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape.Nature. 2019; 573: 281-286Crossref PubMed Scopus (159) Google Scholar,33Baubec T. Colombo D.F. Wirbelauer C. et al.Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.Nature. 2015; 520: 243-247Crossref PubMed Scopus (396) Google Scholar (Figure 1C). DNA methylation and H3K36me2/3 modifications associated with active gene bodies and noncoding regions of euchromatin are positively correlated: H3K36me2/3 mediates recruitment of de novo DNA methyltransferase DNMT3A through direct binding in ESCs, as well as in head and neck squamous cell carcinoma,32Weinberg D.N. Papillon-Cavanagh S. Chen H. et al.The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape.Nature. 2019; 573: 281-286Crossref PubMed Scopus (159) Google Scholar,33Baubec T. Colombo D.F. Wirbelauer C. et al.Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.Nature. 2015; 520: 243-247Crossref PubMed Scopus (396) Google Scholar where changes in H3K36 methylation patterns induce flux in DNA methylation. Sequential chromatin immunoprecipitation–bisulfite assays show increased H3K27me3 occupancy in the absence of DNMTs at CpG-dense regions hypermethylated in ESCs.31Brinkman A.B. Gu H. Bartels S.J. et al.Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk.Genome Res. 2012; 22: 1128-1138Crossref PubMed Scopus (270) Google Scholar However, expected H3K27me3 and DNA methylation antagonism is not obvious when the relationship between H3K27me3 and DNA methylation is examined globally, as reported across 111 human reference epigenome profiles.19Kassis J.A. Brown J.L. Polycomb group response elements in Drosophila and vertebrates.Adv Genet. 2013; 81: 83-118Crossref PubMed Scopus (139) Google Scholar From a genome-wide vantage point, considering CGIs and non-CGI regions of the genome, H3K27me3 enrichment is positively correlated with DNA methylation in most cells; an observation that is concurred multiple times in human ESCs.21Lorzadeh A. Bilenky M. Hammond C. et al.Nucleosome density ChIP-seq identifies distinct chromatin modification signatures associated with MNase accessibility.Cell Rep. 2016; 17: 2112-2124Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,31Brinkman A.B. Gu H. Bartels S.J. et al.Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk.Genome Res. 2012; 22: 1128-1138Crossref PubMed Scopus (270) Google Scholar This contradictory relationship suggests that the association between DNA methylation and H3K27me3 is highly dependent on CpG density and genomic features (eg, CGIs and promoters),21Lorzadeh A. Bilenky M. Hammond C. et al.Nucleosome density ChIP-seq identifies distinct chromatin modification signatures associated with MNase accessibility.Cell Rep. 2016; 17: 2112-2124Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar,24Kundaje A. Meuleman W. Ernst J. et al.Integrative analysis of 111 reference human epigenomes.Nature. 2015; 518: 317-330Crossref PubMed Scopus (3337) Google Scholar,29Neri F. Krepelova A. Incarnato D. et al.Dnmt3L antagonizes DNA methylation at bivalent promoters and favors DNA methylation at gene bodies in ESCs.Cell. 2013; 155: 121-134Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar,31Brinkman A.B. Gu H. Bartels S.J. et al.Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk.Genome Res. 2012; 22: 1128-1138Crossref PubMed Scopus (270) Google Scholar where H3K27me3 and DNA methylation are antagonistic at CpG-dense regions (ie, CGIs) and co-occur at orphan CpGs or CpG poor regions. These observations put forward the importance of other epigenetic layers and complexes, which act to selectively recruit or block either polycomb complexes or DNMTs. Moreover, they call to attention that disruption in either DNA methylation or H3K27me3 distorts the landscape of the other. Together, these findings emphasize that understanding the mechanistic interdependence of various chromatin layers, particularly as manifested in developmental and homeostatic processes, has significant implications for precisely investigating the failure of this machinery in CRC. The majority of CRCs show a well-defined mutational progression in genes, such as the APC, KRAS, and TP53.34Fearon E.R. Vogelstein B. A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9812) Google Scholar Although the effect of DNA methylation changes on CRC is well studied, the role of PRC2 in CRC may be profoundly linked to DNA methylation rather than mutation-induced changes to its function, as observed in other cancers. We start by highlighting a few such cases that are pertinent to understanding the role of PRC2 in CRC, which is detailed in the following section. Deregulation and/or mutation of various PRC2 components has been observed in numerous diseases, including cancers of blood, prostate, brain, and gastrointestinal tissues.35McCabe M.T. Ott H.M. Ganji G. et al.EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations.Nature. 2012; 492: 108-812Crossref PubMed Scopus (1286) Google Scholar, 36Jain P. Di Croce L. Mutations and deletions of PRC2 in prostate cancer.Bioessays. 2016; 38: 446-454Crossref PubMed Scopus (12) Google Scholar, 37Cooney E. Bi W. Schlesinger A.E. et al.Novel EED mutation in patient with Weaver syndrome.Am J Med Genet A. 2017; 173: 541-545Crossref PubMed Scopus (36) Google Scholar, 38Zhang M. Wang Y. Jones S. et al.Somatic mutations of SUZ12 in malignant peripheral nerve sheath tumors.Nat Genet. 2014; 46: 1170-1172Crossref PubMed Scopus (173) Google Scholar, 39Ueda T. Sanada M. Matsui H. et al.EED mutants impair polycomb repressive complex 2 in myelodysplastic syndrome and related neoplasms.Leukemia. 2012; 26 (2557–2260)Crossref PubMed Scopus (34) Google Scholar, 40Morin R.D. Johnson N.A. Severson T.M. et al.Somatic mutations a