Navigating the DNA methylation landscape of cancer
Authors: Atsuya Nishiyama, Makoto Nakanishi
Journal: Trends in Genetics (2021)

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. Genet. 1999; 22: 203-206Crossref PubMed Scopus (268) Google Scholar], genomic imprinting [4.Li E. et al.Role for DNA methylation in genomic imprinting.Nature. 1993; 366: 362-365Crossref PubMed Scopus (1684) Google Scholar, 5.Stöger R. et al.Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal.Cell. 1993; 73: 61-71Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 6.Ferguson-Smith A.C. et al.Parental-origin-specific epigenetic modification of the mouse H19 gene.Nature. 1993; 362: 751-755Crossref PubMed Google Scholar], X chromosome inactivation [7.Mohandas T. et al.Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation.Science. 1981; 211: 393-396Crossref PubMed Google Scholar,8.Wolf S.F. et al.Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation.Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2806-2810Crossref PubMed Google Scholar], transposon (see Glossary) silencing [9.Walsh C.P. et al.Transcription of IAP endogenous retroviruses is constrained by cytosine methylation.Nat. Genet. 1998; 20: 116-117Crossref PubMed Scopus (808) Google Scholar], and genome stability [10.Eden A. et al.Chromosomal instability and tumors promoted by DNA hypomethylation.Science. 2003; 300: 455Crossref PubMed Scopus (1008) Google Scholar,11.Karpf A.R. Matsui S. Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells.Cancer Res. 2005; 65: 8635-8639Crossref PubMed Scopus (190) Google Scholar]. DNA methylation is mainly catalyzed by three enzymes, DNMT1, DNMT3A, and DNMT3B (Figure 1). 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]. In contrast, DNA methylation is not necessarily required in embryonic stem (ES) cells; even when CpG methylation is completely lost by combined KO of three DNMTs Dnmt1, Dnmt3a, and Dnmt3b, there is a minimal change in phenotype in undifferentiated ES cells [14.Tsumura A. et al.Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b.Genes Cells. 2006; 11: 805-814Crossref PubMed Scopus (380) Google Scholar]. Although DNA methylation is a stable modification, there are also several pathways of demethylation and these pathways play an important role in the regulation of DNA methylation in various biological contexts (Box 3). 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. Genet. 2014; 46: 17-23Crossref PubMed Scopus (203) Google Scholar] or DNA methylation valleys [19.Xie W. et al.Epigenomic analysis of multilineage differentiation of human embryonic stem cells.Cell. 2013; 153: 1134-1148Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar] that are associated with either the active histone mark H3K4me3 or the inhibitory mark H3K27me3.Figure 2Regulatory mechanism of DNMT3A activity and localization.Show full caption(A) In the absence of histone H3, DNMT3A exists in an autoinhibitory state, in which the ATRX-DNMT3A-DNMT3L (ADD) domain interacts with the catalytic domain (CD) to inhibit DNA binding. H3K4me0 interacts with the ADD domain to break the autoinhibition and activate DNMT3A. H3K4me0 interacts with the ADD domain to release autoinhibition and activate DNMT3A. H3K36me3 interacts with the Pro-Trp-Trp-Pro (PWWP) domain of DNMT3A to promote DNA methylation in the gene body. 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]. DNMT3 family proteins are composed of a Pro-Trp-Trp-Pro (PWWP) domain at the N terminal region, an ATRX-DNMT3A-DNMT3L (ADD) domain in the central region, and an enzyme domain at the C terminal region (Figure 1) [126.Goll M.G. Bestor T.H. Eukaryotic cytosine methyltransferases.Annu. Rev. Biochem. 2005; 74: 481-514Crossref PubMed Scopus (1492) Google Scholar]. Both the PWWP and ADD domains play important roles in the localization of DNMT3A and DNMT3B to their target sites and in the regulation of enzymatic activity. The PWWP domain is required for proper chromatin binding of DNMT3A and DNMT3B [127.Ge Y.-Z. et al.Chromatin targeting of de novo DNA methyltransferases by the PWWP domain.J. Biol. Chem. 2004; 279: 25447-25454Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar] and acts as a reader module to recognize and bind histones H3K36me2 and H3K36me3 [128.Dhayalan A. et al.The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation.J. Biol. Chem. 2010; 285: 26114-26120Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar]. Histone H3K36me3 enriches the gene body of actively transcribed genes by undergoing catalysis by the SETD2 protein (also called RNAPII subunit B1), which binds to the C terminal domain of RNA polymerase II [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, 130.Kizer K.O. et al.A novel domain in Set2 mediates RNA polymerase II interaction and couples histone H3 K36 methylation with transcript elongation.Mol. Cell. Biol. 2005; 25: 3305-3316Crossref PubMed Scopus (329) Google Scholar, 131.Krogan N.J. et al.Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II.Mol. Cell. Biol. 2003; 23: 4207-4218Crossref PubMed Scopus (492) Google Scholar]. 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]. The crystal structure of the ADD domain of DNMT3A in complex with the amino-terminal tail of histone H3 showed the specific interaction of the ADD domain with H3K4me0, not with H3K4me2 and H3K4me3 [137.Otani J. et al.Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain.EMBO Rep. 2009; 10: 1235-1241Crossref PubMed Scopus (250) Google Scholar, 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, 139.Noh K.-M. et al.Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape and phenotypic features of mouse ESCs.Mol. Cell. 2015; 59: 89-103Abstract Full Text Full Text PDF PubMed Google Scholar]. These results are consistent with the fact that H3K4me3 and DNA methylation distribution are mutually exclusive [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, 139.Noh K.-M. et al.Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape and phenotypic features of mouse ESCs.Mol. Cell. 2015; 59: 89-103Abstract Full Text Full Text PDF PubMed Google Scholar, 140.Zhang Y. et al.Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail.Nucleic Acids Res. 2010; 38: 4246-4253Crossref PubMed Scopus (243) Google Scholar, 141.Li B.-Z. et al.Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase.Cell Res. 2011; 21: 1172-1181Crossref PubMed Scopus (81) Google Scholar, 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]. Moreover, the histone H3 tail stimulates the enzymatic activity of DNMT3A in vitro [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,140.Zhang Y. et al.Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail.Nucleic Acids Res. 2010; 38: 4246-4253Crossref PubMed Scopus (243) Google Scholar,141.Li B.-Z. et al.Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase.Cell Res. 2011; 21: 1172-1181Crossref PubMed Scopus (81) Google Scholar]. In the absence of the histone H3 tail, DNMT3A exists in an autoinhibitory form in which the ADD domain directly inhibits DNA binding of the catalytic domain (CD). 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]. Ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), also called ICPB90 in humans and Np95 in mice, plays a critical role in maintenance of DNA methylation [116.Sharif J. et al.The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA.Nature. 2007; 450: 908-912Crossref PubMed Scopus (830) Google Scholar,117.Bostick M. et al.UHRF1 Plays a role in maintaining DNA methylation in mammalian cells.Science. 2007; 317: 1760-1764Crossref PubMed Scopus (867) Google Scholar]. UHRF1 binds to hemi-methylated DNA via its SET- and RING-associated (SRA) domain [146.Arita K. et al.Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism.Nature. 2008; 455: 818-821Crossref PubMed Scopus (335) Google Scholar, 147.Hashimoto H. et al.The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix.Nature. 2008; 455: 826-829Crossref PubMed Scopus (300) Google Scholar, 148.Avvakumov G.V. et al.Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1.Nature. 2008; 455: 822-825Crossref PubMed Scopus (331) Google Scholar] and accumulates at DNA replication sites to recruit DNMT1 [116.Sharif J. et al.The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA.Nature. 2007; 450: 908-912Crossref PubMed Scopus (830) Google Scholar,117.Bostick M. et al.UHRF1 Plays a role in maintaining DNA methylation in mammalian cells.Science. 2007; 317: 1760-1764Crossref PubMed Scopus (867) Google Scholar]. Recent studies have indicated that an E3 ubiquitin ligase activity of UHRF1 is critical for efficient maintenance of DNA methylation. Upon SRA-mediated binding to hemi-methylated DNA, UHRF1 mono-ubiquitylates Lys14-, Lys18, and/or Lys 23 in histone H3 [115.Nishiyama A. et al.Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication.Nature. 2013; 502: 249-253Crossref PubMed Scopus (206) Google Scholar,149.Ishiyama S. et al.Structure of the Dnmt1 reader module complexed with a unique two-mono-ubiquitin mark on histone H3 reveals the basis for DNA methylation maintenance.Mol. Cell. 2017; 68: 350-360Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,150.Qin W. et al.DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.Cell Res. 2015; 25: 911-929Crossref PubMed Scopus (127) Google Scholar]. Hemi-methyl-DNA binding stimulates UHRF1 E3 activity and/or accessibility to histone H3 in vitro [120.Harrison J.S. et al.Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.eLife. 2016; 5e17101Crossref PubMed Scopus (64) Google Scholar,151.Fang J. et al.Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition.Nat. Commun. 2016; 7: 11197Crossref PubMed Scopus (66) Google Scholar]. The N terminal Ubiquitin-like (UBL) domain also contributes to UHRF1 E3 ligase activity via interaction with the E2 ubiquitin-conjugating enzyme Ubch5a/UBE2D1 [123.Foster B.M. et al.Critical role of the UBL domain in stimulating the E3 ubiquitin ligase activity of UHRF1 toward chromatin.Mol. Cell. 2018; 72: 739-752Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar,124.DaRosa P.A. et al.A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation.Mol. Cell. 2018; 72: 753-765Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar]. Multiple mono-ubiquitylated histone H3 is in turn recognized and bound by DNMT1 through two ubiquitin binding modules in DNMT1-RFTS [118.Li T. et al.Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation.Nucleic Acids Res. 2018; 46: 3218-3231Crossref PubMed Scopus (53) Google Scholar,149.Ishiyama S. et al.Structure of the Dnmt1 reader module complexed with a unique two-mono-ubiquitin mark on histone H3 reveals the basis for DNA methylation maintenance.Mol. Cell. 2017; 68: 350-360Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar,150.Qin W. et al.DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.Cell Res. 2015; 25: 911-929Crossref PubMed Scopus (127) Google Scholar], which was previously shown to be essential for DNMT1 recruitment to DNA replication sites [152.Leonhardt H. et al.A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei.Cell. 1992; 71: 865-873Abstract Full Text PDF PubMed Scopus (784) Google Scholar]. The RFTS domain is also proposed to function as an autoinhibitory module via interaction with the catalytic pocket of DNMT1 [153.Takeshita K. et al.Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1).Proc. Natl. Acad. Sci. 2011; 108: 9055-9059Crossref PubMed Scopus (0) Google Scholar,154.Syeda F. et al.The replication focus targeting sequence (RFTS) domain is a DNA-competitive inhibitor of Dnmt1.J. Biol. Chem. 2011; 286: 15344-15351Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Importantly, DNMT1 binding to dual mono-ubiquitylated H3 enhances DNMT1 methyltransferase activity [149.Ishiyama S. et al.Structure of the Dnmt1 reader module complexed with a unique two-mono-ubiquitin mark on histone H3 reveals the basis for DNA methylation maintenance.Mol. Cell. 2017; 68: 350-360Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar], presumably via a gross conformational change and opening of its active site. 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. Commun. 2020; 11: 1222Crossref PubMed Scopus (25) Google Scholar], suggesting that the PHD finger of UHRF1 is critical for recognizing the N terminal end of its substrate proteins. During S phase, PAF15 undergoes UHRF1-dependent dual mono-ubiquitylation on two evolutionarily conserved lysine residues in a DNA replication-coupled manner and interacts with DNMT1 [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]. Importantly, the PAF15 interaction with PCNA is essential for PAF15 function in the maintenance of DNA methylation. 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]. DNA demethylation is particularly important in preimplantation embryonic and primordial germ cell (PGC) development, as it is active in setting up pluripotent states in early embryos and in erasing parental-origin-specific imprints in developing PGCs [159.Lee J. et al.Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells.Development. 2002; 129: 1807-1817Crossref PubMed Scopus (50) Google Scholar,160.Hill P. et al.DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: an emerging complex story.Genomics. 2014; 104: 324-333Crossref PubMed Scopus (101) Google Scholar]. DNA demethylation occurs mainly by two different pathways: (i) DNA replication-dependent dilution of DNA methylation marks by inactivation of de novo DNA methylation or the maintenance DNA methylation machinery [161.Kagiwada S. et al.Replication-coupled passive DNA demethylation for the erasure of genome imprints in mice.EMBO J. 2013; 32: 340-353Crossref PubMed Scopus (197) Google Scholar, 162.Ohno R. et al.A replication-dependent passive mechanism modulates DNA demethylation in mouse primordial germ cells.Development. 2013; 140: 2892-2903Crossref PubMed Scopus (52) Google Scholar, 163.Seisenberger S. et al.The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells.Mol. Cell. 2012; 48: 849-862Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 164.Kurimoto K. et al.Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice.Gene Dev. 2008; 22: 1617-1635Crossref PubMed Scopus (0) Google Scholar], and (ii) Ten-eleven Translocation (TET)-dependent active demethylation [165.Yamaguchi S. et al.Role of Tet1 in erasure of genomic imprinting.Nature. 2013; 504: 460-464Crossref PubMed Scopus (145) Google Scholar, 166.Yamaguchi S. et al.Tet1 controls meiosis by regulating meiotic gene expression.Nature. 2012; 492: 443-447Crossref PubMed Scopus (182) Google Scholar, 167.Wu H. Zhang Y. Reversing DNA methylati