Navigating the DNA methylation landscape of cancer

DNA methylation is an important epigenetic modification that defines the properties of cells. Genome-wide hypomethylation, as well as hypermethylation of CpG islands associated with tumor suppressor genes and developmental regulators, are characteristics of cancer cells.DNA methyltransferases normally exist in an inactive form and their localization and activation are regulated by interaction with unique histone modifications at DNA methylation sites.Changes in DNA methylation patterns associated with carcinogenesis progress gradually with cell proliferation. Genome-wide hypomethylation is found in DNA blocks called partially methylated domains (PMDs) and it frequently occurs in solo-WCGW sequences that have no nearby CpG sequences and are adjacent to A or C.CpG island methylation primarily targets promoters characterized by low gene expression marked by H3K27m3, with the replacement of histone modifications by DNA methylation ensuring more stable gene repression. DNA methylation is a chemical modification that defines cell type and lineage through the control of gene expression and genome stability. Disruption of DNA methylation control mechanisms causes a variety of diseases, including cancer. Cancer cells are characterized by aberrant DNA methylation (i.e., genome-wide hypomethylation and site-specific hypermethylation), mainly targeting CpG islands in gene expression regulatory elements. In particular, the early findings that a variety of tumor suppressor genes (TSGs) are targets of DNA hypermethylation in cancer led to the proposal of a model in which aberrant DNA methylation promotes cellular oncogenesis through TSGs silencing. However, recent genome-wide analyses have revealed that this classical model needs to be reconsidered. In this review, we will discuss the molecular mechanisms of DNA methylation abnormalities in cancer as well as their therapeutic potential. DNA methylation is a chemical modification that defines cell type and lineage through the control of gene expression and genome stability. Disruption of DNA methylation control mechanisms causes a variety of diseases, including cancer. Cancer cells are characterized by aberrant DNA methylation (i.e., genome-wide hypomethylation and site-specific hypermethylation), mainly targeting CpG islands in gene expression regulatory elements. In particular, the early findings that a variety of tumor suppressor genes (TSGs) are targets of DNA hypermethylation in cancer led to the proposal of a model in which aberrant DNA methylation promotes cellular oncogenesis through TSGs silencing. However, recent genome-wide analyses have revealed that this classical model needs to be reconsidered. In this review, we will discuss the molecular mechanisms of DNA methylation abnormalities in cancer as well as their therapeutic potential. DNA methylation is a chemical modification that plays an important role in the regulation of epigenetic gene expression [1.Yisraeli J. et al.Muscle-specific activation of a methylated chimeric actin gene.Cell. 1986; 46: 409-416Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 2.Busslinger M. et al.DNA methylation and the regulation of globin gene expression.Cell. 1983; 34: 197-206Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 3.Siegfried Z. et al.DNA methylation represses transcription in vivo.Nat. 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De novo DNA methylation is mainly catalyzed by the DNA methyltransferases (DNMTs) DNMT3A and 3B (Box 1), while established DNA methylation patterns are maintained by the daughter DNA through a maintenance DNA methylation mechanism during cell proliferation (Box 2). Both de novo DNA methylation and maintenance DNA methylation are important for normal development. DNMT1 inactivation and DNMT3A/3B double knockout (KO) mouse embryos show significant growth inhibition and are lethal before mid-gestation [12.Li E. et al.Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.Cell. 1992; 69: 915-926Abstract Full Text PDF PubMed Scopus (3073) Google Scholar,13.Okano M. et al.DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4122) Google Scholar]. 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In normal cells, most CpG sequences in the genome are methylated, but CpG islands and the nearby CpG island shores (the region within 2 kb of the islands) are exceptionally hypomethylated [15.Irizarry R.A. et al.The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores.Nat. Genet. 2009; 41: 178-186Crossref PubMed Scopus (1528) Google Scholar,16.Doi A. et al.Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.Nat. Genet. 2009; 41: 1350-1353Crossref PubMed Scopus (892) Google Scholar]. Many of these hypomethylated regions of DNA function as elements that regulate gene expression, such as promoters and enhancers. In addition, systematic analysis of unmethylated DNA and methylated CpG as ligands has revealed that DNA methylation promotes the binding of many transcription factors [17.Yin Y. et al.Impact of cytosine methylation on DNA binding specificities of human transcription factors.Science. 2017; 356eaaj2239Crossref PubMed Scopus (373) Google Scholar]. Recently, broad unmethylated regions were also reported as DNA methylation canyons [18.Jeong M. et al.Large conserved domains of low DNA methylation maintained by Dnmt3a.Nat. 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It has also been reported that H3K36Kme2 similarly regulates the localization of DNMT3B to intergenic regions through binding to the PWWP domain. (B) DNMT3A2 and DNMT3B3, catalytically inactive regulatory subunits, form heterotetrameric 3B3-3A2-3A2-3B3. One of the two catalytic-like domains of DNMT3B3 interacts with the acidic patch of the nucleosome core particle, leading to the binding of two DNMT3A2 to the linker DNA. The interaction of DNMT3B3 with the acidic patch is also important for chromatin binding of DNA methyltransferase (DNMT) complexes and de novo methylation, indicating the importance of the acidic patch, in addition to H3K4me0 and H3K36me2 and me3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Box 1De novo DNA methylationMammalian DNA methylation at CpG sites is mainly established by the de novo methyltransferases, DNMT3A and DNMT3B [13.Okano M. et al.DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4122) Google Scholar,125.Okano M. et al.Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases.Nat. Genet. 1998; 19: 219-220Crossref PubMed Scopus (1206) Google Scholar]. 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In mouse ES cells, localization of DNMT3B to the gene body occurs through binding to H3K36me3 with the PWWP domain [132.Baubec T. et al.Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.Nature. 2015; 520: 243-247Crossref PubMed Scopus (350) Google Scholar]. Transcription- and H3K36me3-mediated de novo DNA methylation is also important for the establishment of the oocyte methylome [133.Xu Q. et al.SETD2 regulates the maternal epigenome, genomic imprinting and embryonic development.Nat. Genet. 2019; 51: 844-856Crossref PubMed Scopus (64) Google Scholar]. Several recent studies have shown that H3K36me2 regulates DNA methylation differently. NSD1 and NSD2 are methyltransferases that regulate K36me2 of histone H3 [129.Wagner E.J. Carpenter P.B. Understanding the language of Lys36 methylation at histone H3.Nat. Rev. Mol. Cell Biol. 2012; 13: 115-126Crossref PubMed Scopus (534) Google Scholar]. Genome-wide analysis using mouse mesenchymal stem cells (MSCs) showed that DNMT3A localization to intergenic regions depends on H3K36me2 in MSCs [134.Weinberg D.N. et al.The histone mark H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape.Nature. 2019; 573: 281-286Crossref PubMed Scopus (97) Google Scholar]. NSD1-mediated de novo methylation has also recently been reported in male mouse germlines [135.Shirane K. et al.NSD1-deposited H3K36me2 directs de novo methylation in the mouse male germline and counteracts Polycomb-associated silencing.Nat. Genet. 2020; 52: 1088-1098Crossref PubMed Scopus (15) Google Scholar]. Thus, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies may specify the localization of DNMT3A and DNMT3B, respectively, to these regions through interaction with the PWWP domain.The ADD domain acts as a molecular module that recognizes unmodified histone H3 lysine 4 [136.Ooi S.K. et al.DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA.Nature. 2007; 448: 714-717Crossref PubMed Scopus (1040) Google Scholar]. 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The binding of H3K4me0, but not H3K4me3, with the ADD domain disrupts the ADD-CD interaction and cancels autoinhibition of DNMT3A (Figure 2A) [138.Guo X. et al.Structural insight into autoinhibition and histone H3-induced activation of DNMT3A.Nature. 2015; 517: 640-644Crossref PubMed Scopus (188) Google Scholar]. This mechanism would ensure the suppression of unfavorable DNA methylation at the H3K4me3-marked promoter, even when DNMT3A is recruited.Figure 3Ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1)-dependent ubiquitin signaling orchestrates maintenance of DNA methylation.Show full captionDuring DNA replication, methylated DNA is converted to hemi-methylated DNA in which only the parent strand is methylated. UHRF1 specifically binds to hemi-methylated DNA and targets two conserved Lys residues at the N terminus of PCNA-associated factor 15 (PAF15) for dual monoubiquitylation. Ubiquitinated PAF15 promotes the localization and activation of DNMT1 at DNA methylation sites through specific interaction with the RFTS domain of DNMT1. Hemi-methylated DNA left after passage through the replication machinery is converted to fully methylated DNA, probably via UHRF1-dependent dual monoubiquitination of histone H3. The interaction between DNMT1 and USP7 indicates that USP7 may act as a deubiquitinating enzyme that antagonizes UHRF1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Box 2Maintenance of DNA methylationProper DNMT1 localization and activation at sites of DNA methylation require various regulatory factors. Early studies demonstrated that DNMT1 interacts and colocalizes with PCNA at replication foci [143.Chuang L. et al.Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1.Science. 1997; 277: 1996-2000Crossref PubMed Scopus (755) Google Scholar], but later it was found that the DNMT1/PCNA interaction is not essential for maintenance of DNA methylation [144.Spada F. et al.DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells.J. Cell Biol. 2007; 176: 565-571Crossref PubMed Scopus (145) Google Scholar,145.Easwaran H.P. et al.Replication-independent chromatin loading of Dnmt1 during G2 and M phases.EMBO Rep. 2004; 5: 1181-1186Crossref PubMed Scopus (130) Google Scholar]. 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These data indicate that ubiquitylated histone H3 acts as a platform to recruit and activate DNMT1.Recently, PCNA-associated factor 15 (PAF15) has emerged as an another substrate for UHRF1-dependent multiple mono-ubiquitylation [121.Nishiyama A. et al.Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.Nat. Commun. 2020; 11: 1222Crossref PubMed Scopus (25) Google Scholar,155.Karg E. et al.Ubiquitome analysis reveals PCNA-associated factor 15 (PAF15) as a specific ubiquitination target of UHRF1 in embryonic stem cells.J. Mol. Biol. 2017; 429: 3814-3824Crossref PubMed Scopus (19) Google Scholar]. The PHD of UHRF1 specifically recognizes and binds to the sequence 2VRTK5, at the extreme N terminus of PAF15, in a manner similar to that of the ARTK of histone H3 [121.Nishiyama A. et al.Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.Nat. 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PAF15 binds to PCNA at the front face and the N terminus of PAF15 interacts with the inner ring of PCNA and exits the clamp from the back face [156.March M. et al.p15PAF binding to PCNA modulates the DNA sliding surface.Nucleic Acids Res. 2018; 46: 9816-9828Crossref PubMed Scopus (7) Google Scholar], suggesting that the PAF15 ubiquitylation could directly recruit DNMT1 to the back face of PCNA, facilitating the maintenance of DNA methylation (Figure 3).Box 3DNA demethylationDuring development, the DNA methylation pattern fluctuates dynamically through genome-wide DNA demethylation and de novo methylation [142.Guibert S. et al.Global profiling of DNA methylation erasure in mouse primordial germ cells.Genome Res. 2012; 22: 633-641Crossref PubMed Scopus (224) Google Scholar,157.Hajkova P. et al.Chromatin dynamics during epigenetic reprogramming in the mouse germ line.Nature. 2008; 452: 877-881Crossref PubMed Scopus (482) Google Scholar,158.Hackett J.A. et al.Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine.Science. 2013; 339: 448-452Crossref PubMed Scopus (511) Google Scholar]. 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