Molecular coupling of DNA methylation and histone methylation

Abstract

The combinatorial pattern of DNA and histone modifications constitutes an epigenetic ‘code’ that shapes gene-expression patterns by enabling or restricting the transcriptional potential of genomic domains. DNA methylation is associated with histone modifications, particularly the absence of histone H3 lysine 4 methylation (H3K4me0) and the presence of H3K9 methylation. This article focuses on three protein domains (ATRX–Dnmt3–Dnmt3L [ADD], Cys–X–X–Cys [CXXC] and the methyl-CpG-binding domain [MBD]) and the functional implications of domain architecture in the mechanisms linking histone methylation and DNA methylation in mammalian cells. The DNA methyltransferase DNMT3a and its accessory protein DNMT3L contain a H3K4me0-interacting ADD domain that links the DNA methylation reaction with unmodified H3K4. The H3K4 methyltransferase MLL1 contains a CpG-interacting CXXC domain that may couple the H3K4 methylation reaction to unmethylated DNA. Another H3K4 methyltransferase, SET1, although lacking an intrinsic CXXC domain, interacts directly with an accessory protein CFP1 that contains the same domain. The H3K9 methyltransferase SETDB1 contains a putative MBD that potentially links the H3K4 methylation reaction to methylated DNA or may do so through the interaction with the MBD containing protein MBD1. Finally, we consider the domain structure of the DNA methyltransferase DNMT1, its accessory protein UHRF1 and their associated proteins, and propose a mechanism by which DNA methylation and histone methylation may be coordinately maintained through mitotic cell division, allowing for the transmission of parental DNA and for the histone methylation patterns to be copied to newly replicated chromatin.

Epigenetics, the cell-type-specific interpretation of genetic material, relies on DNA methylation and post-translational histone modifications to regulate gene function. Nucleosomes, the fundamental building unit that packages DNA, consist of approximately 146 bp of DNA wrapped approximately 1.8-times around a histone octamer that is evolutionarily conserved [1]. Histones are subject to considerable post-translational modifications including acetylation, methylation, ubiquitylation and sumoylation of lysine residues and methylation of arginine residues [2]. The combinatorial pattern of DNA and histone modifications constitutes an epigenetic ‘code’ that shapes gene-expression patterns by enabling or restricting the transcriptional potential of genomic domains. The code is written by sequence and site-specific modification enzymes and interpreted by effector molecules that mediate the assembly of higher-order chromatin structures. Epigenetic regulation plays a fundamental role in gene expression [3], DNA replication [4] and, recombination and repair [5], and is responsible for stem cell development [6] and cellular differentiation [7]. Moreover, alterations in epigenetic modifications accompany the aging process and contribute to the pathogenesis of cancer [8–14] and degenerative diseases [15,16].

One broad theme that has emerged is that a web of interactions tightly coordinates the modification of a segment of DNA and its associated histones, particularly histone H3. This article focuses on three protein domains (ATRX–Dnmt3–Dnmt3L [ADD], Cys–X–X–Cys [CXXC] and the methyl-CpG-binding domain [MBD]) and how they characterize the functional links between histone and DNA modification in mammalian cells. In particular we consider the relationship between DNA CpG methylation and histone H3 methylation on lysines 4 and 9 [17–23]. DNA methylation and histone lysine methylation are intimately connected with one another [17,19–21]. In fact, genome-scale DNA methylation profiles suggest that DNA methylation is correlated to histone methylation patterns [18]. Specifically, DNA methylation is associated with the absence of H3K4 methylation (H3K4me0) and the presence of H3K9 methylation, but has little correlation with methylation of H3K27 [22]. In vivo studies support a molecular link between the mechanisms that maintain DNA methylation and H3K9 methylation. Studies in Neurospora and Arabidopsis have demonstrated a strict dependence of DNA methylation on the H3K9 methyltransferases Dim-5 and KRYPTONITE [24–26]. The relationship in mammals appears more complex, however, as deletion of the H3K9 trimethyltransferases SUV39H1/H2 [27] or SETDB1 [28] has only a minor impact on DNA methylation at constitutive heterochromatin or endogenous retroelements, respectively. That the mechanisms defining genomic patterns of DNA methylation, H3K4 methylation and H3K9 methylation are functionally linked is underscored by the finding that treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine leads to depletion of DNA methylation and a loss of H3K9 methylation and a corresponding increase in H3K4 methylation [29].

ADD domains of Dnmt3L/3a/3b link DNA methylation to unmethylated H3K4

DNA methylation in mammals is coordinately established and maintained by two DNA methyltransferase families, the so-called ‘de novo’ methyltransferases of the Dnmt3 family and the ‘maintenance’ methyltransferase Dnmt1. The Dnmt3 family includes two active de novo Dnmts, Dnmt3a and -3b, and one regulatory factor, Dnmt3L (Figure 1A). Dnmt3a and -3b have similar domain arrangements: both contain a variable region at the N-terminus, followed by a Pro–Trp–Trp–Pro domain, an ADD domain and a C-terminal catalytic domain. The amino acid sequence of Dnmt3L is very similar to that of Dnmt3a and -3b in the ADD domain (Figure 1B). Dnmt3L associates in vivo with not only Dnmt3b and -3a2 (a shorter isoform of Dnmt3a) [30], but also with the four core histones [31]. Peptide interaction assays and cocrystallization of Dnmt3L with the amino tail of H3 demonstrated that Dnmt3L specifically interacts with the amino terminus of histone H3 only when H3K4 is not modified [31]. These data suggest that Dnmt3L acts as a sensor for H3K4 methylation; when methylation is absent, Dnmt3L induces de novo DNA methylation by docking Dnmt3a to the nucleosome (Figure 1C).

Figure 1. H3K4me0-interacting proteins.

(A) Domain architecture of known H3K4me0-interacting proteins containing either ADD domain or PHD finger. ATRX interacts with histone H3.3. (B) Sequence alignment of ADD domains and PHD fingers known to interact with H3K4me0. (C) Model of the reactions that regulate DNA methylation by Dnmt3a/3L. Recognition of H3K4me0 by the ADD domain of DNMT3 directs the DNA methylation reaction. The ‘m’ in a circle indicates one or more methyl groups in DNA (5-methyl-cytosine) or histone lysines (methylated lysine). (D) Structure of the ADD domain of Dnmt3a (PDB ID: 3A1B) showing the interaction with the histone H3 tail.

ADD: ATRX–Dnmt3–Dnmt3L; Chromo: Chromosome; PDB: Protein database; PHD: Plant homeodomain; RING: Really interesting new gene; SRA: SET and RING associated.

Histone–Dnmt3L–Dnmt3a–DNA interactions have recently been studied in the budding yeast Saccharomyces cerevisiae [32], which has no detectable DNA methylation [33] and lacks Dnmt orthologs. Introduction of the murine methyltransferase Dnmt1 or -3a in yeast leads to detectable, but extremely low levels of DNA methylation [34]. By contrast, a substantially higher level of de novo methylation could be achieved in yeast by coexpressing murine Dnmt3a and -3L [32]. This induced DNA methylation was found preferentially in heterochromatic regions where H3K4 methylation is rare. When genes for components of the H3K4-methylating complex COMPASS/Set1 were disrupted in the context of Dnmt3a/3L overexpression, a greater level of genomic DNA methylation was observed. Deletions or targeted mutations in the ADD domain of Dnmt3L (Figure 1B) inhibited both global DNA methylation and the ability of Dnmt3L to associate with a H3K4me0 peptide. These same Dnmt3L mutants failed to restore normal DNA methylation to a specific promoter when introduced into embryonic stem cells from Dnmt3L^-/- mice [32]. That H3K4 methylation status plays an important role in the establishment of DNA methylation is further supported by the finding that mammalian LSD1 and LSD2, two related lysine-specific demethylases whose substrates include mono- and di-methylated H3K4 (H3K4me1/2), are absolutely essential in maintaining global DNA methylation [35], or establishing maternal DNA genomic imprints [36], respectively. Indeed, disruption of LSD1 results in earlier embryonic lethality and a more severe hypomethylation defect than disruption of the Dnmts themselves [35].

The aforementioned data lead to a model in which Dnmt3L binds to H3K4me0 (via its ADD domain) and recruits Dnmt3a to regions of chromatin where H3K4 is unmethylated. Such a model could explain part of the puzzle of how DNA methylation patterns are established de novo during embryonic and germ cell development, when both proteins are expressed [37]. However, whereas Dnmt3a and -3b expression is retained in somatic cells, Dnmt3L is expressed poorly if at all in differentiated cell types. This raises the question of how de novo DNA methylation is restricted in somatic cells, whether Dnmt3a and -3b alone are capable of discriminating H3K4 methylation status, and (if so) the structural basis for that discrimination. To this end, recent work by Jeung et al. demonstrated that in nuclei from HCT116 human colon cancer cells (which do not express DNMT3L), almost all of the cellular DNMT3a and -3b (but not DNMT1) was associated with nucleosomes [38]. Chromatin binding of DNMT3a and -3b required an intact nucleosomal structure, though no other chromatin factors (e.g., HP1 and MBD proteins), suggesting that DNMT3a and -3b alone are capable of direct interaction with chromatin components in addition to DNA. Furthermore, recent in vitro studies indicate that the ADD domains of Dnmt3a or -3b possess the same H3 tail binding specificity as that of Dnmt3L [39]. Indeed, a structure of the Dnmt3a ADD domain in complex with an amino-terminal tail peptide from histone H3 indicates that the ADD domain is sufficient to recognize H3K4me0 (Figure 1D) [40].

The ADD domain consists of 12 cysteines, with the first four forming a 4-Cys zinc finger, and the last eight forming a plant homeodomain (PHD) finger that adopts a ‘cross-braced’ topology of the binding of two Zn atoms (Figure 1B) [41]. The two fingers pack together to form a single globular domain. The two-aspartate motif (DxD) that mediates recognition of H3K4me0 in the structurally characterized Dnmt3a and -3L are located between the two fingers, that is, between the fourth and fifth cysteine (Figure 1B). Interestingly, UHRF1 (ubiquitin-like, containing PHD and really interesting new gene [RING] finger domains 1), a modular multidomain protein important in regulating DNMT1-mediated replication-coupled DNA methlyation [42,43] (reviewed in [44,45]), contains a similar 12-cys architecture (Figure 1B), consisting of a 4-Cys zinc finger and a PHD finger, as illustrated by an NMR structure for the ADD-like domain of human UHRF2 (Protein Data Bank ID 2E6S). The major difference is that the 4-Cys zinc finger and the PHD finger are only one residue apart, and thus lack the H3K4me0-interacting acidic residue(s) between the fourth and fifth cysteine (Figure 1B). Although the UHRF1 ADD-like domain is known to bind the histone H3 tail [46,47], it remains to be seen whether it also senses the methylation state of H3K4.

Another known H3K4me0 binder is the PHD domain of BHC80 [48]. BHC80 (also known as PHF21A) is a component of the LSD1 complex [49]. Like the ADD domain of the Dnmt3 family, the PHD finger of BHC80 binds unmethylated H3K4 and this interaction is specifically abrogated by methylation at this site. Although BHC80 has a conserved DxD motif in its corresponding position (Figure 1B), the PHD domain alone (without the DxD motif) is sufficient to bind H3K4me0 [48]. Asp489 of BHC80, which is located before cysteine 5 but is not part of the DxD motif (Figure 1B), forms an electrostatic interaction with H3K4 [48]. Similarly, the first of two PHD fingers in the autoimmune regulator (AIRE), which also have an aspartate residue in the corresponding position (Asp297), bind to histone H3 tails unmethylated at K4 [50,51].

Another known H3K4me0-specific interaction occurs within the nucleosome remodeling histone deacetylase (NuRD) complex, a multisubunit enzyme complex with roles in both transcriptional regulation and in replication-coupled chromatin assembly [52]. This complex possesses two distinct enzymatic activities that affect chromatin structure and function, an ATP-dependent chromatin remodeling activity (CHD3/4) and histone deacetylase activity (HDAC1/2). The NuRD complex has been demonstrated to bind to histone H3 amino terminal peptides, and this interaction is abrogated by methylation of H3K4 [53,54]. Recently, the NuRD–H3K4me0 interaction has been mapped to CHD4 [55]. CHD4 contains two PHD fingers, the second of which (PHD-2) recognizes the N-terminal tail of histone H3 in an interaction that is inhibited by the methylation of H3K4. Sequence alignment indicates that the first and second PHD fingers of CHD4, as well as that of CHD3, are highly conserved and that the corresponding acidic residues known to be important for H3K4me0 binding in the ADD domain – those N-terminal to cysteine 5 – are also found in the CHD4 PHD-2 (ExD), but not in PHD-1 (Figure 1B). The NuRD complex also contains two members of the MBD protein family, MBD2 and MBD3 (see later). Given that H3K4me0 and methylated CpGs are typically found at sites of repressive transcription, one can speculate that the recognition of these features (H3K4me0 and methyl-CpG) allows for the recruitment and assembly of the NuRD repressor complex at these sites.

CXXC domain links H3K4 methylation to unmethylated CpGs

In humans there are at least eight histone lysine methyltransferases with specificity for H3K4. These include the MLL genes, MLL1–5, hSET1a, hSET1b and ASH1. MLL1/SET1-associated methyltransferase activity appears to be functional only in the context of multiprotein complexes; characterization of these reveals distinct multiprotein complexes for each with several shared components [56]. The MLL family plays an important role in embryonic development and is necessary for the methylation of H3K4 at a subset of genes in the human and mouse genomes, particularly the Hox gene clusters [57]. Translocations involving MLL genes are involved in the etiology of myeloid and lymphoid leukemias. Considering the aforementioned inverse relationship between H3K4 methylation and DNA methylation, it is interesting to note that disruption of the MLL1 gene in mice results in a loss of H3K4 methylation and de novo DNA methylation at some Hox gene promoters [58,59], suggesting that MLL genes directly or indirectly (through H3K4 methylation) prevent DNA methylation or perhaps stabilize unmethylated DNA. In fact, MLL proteins contain a CXXC domain, an evolutionarily conserved domain that mediates selective binding to unmethylated CpGs (Figure 2A) [60–62]. This interaction has now been confirmed by a solution structure of an MLL1-CXXC domain complexed with unmethylated DNA (Figure 2B) [63].

Figure 2. CpG-interacting proteins.

(A) Domain architecture of CXXC domain-containing proteins. Inset: model of the H3K4 methylation reaction by MLL/SET1. CXXC domain-mediated binding to unmethylated CpGs directs the methylation of H3K4 by MLL/SET1 proteins. CXXC4 was initially named as IDAX, and decreased expression of CXXC4 promotes a malignant phenotype in renal cell carcinoma by activating Wnt signaling. CXXC5 (also known as RINF), which was initially identified in a large-scale functional proteomics mapping of human genes, activates the NF-kB and MAPK signaling pathways, and its expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34⁺ progenitors, is required for DNA damage-induced p53 activation and is a BMP4-regulated modulator of Wnt signaling in neural stem cells. (B) Ribbon diagram (top left) and stick diagram (top right) of the MLL1 CXXC domain in complex with unmethylated DNA. Note the recognition of CpG by a Lys–Gln dipeptide in the major groove. Sequence alignment of CXXC domains including candidates from Drosophila and Caenorhabditis elegans (Bottom).

BAH: Bromo-adjacent homology domain; CHD: Chromodomain-helicase-DNA-binding protein; CXXC: Cys–X–X–Cys; DSBH: Double-stranded β-helix; FYRC: FY-rich domain C-terminal region; FYRN: FY-rich domain N-terminal region; IDAX: Inhibiton of the Dvl and Axin complex in the Wnt signaling pathway; MBD: Methyl-CpG-binding domain; PCNA: Proliferating cell nuclear antigen; PHD: Plant homeodomain; RINF: Retinoid-inducible nuclear factor; SET: Su(var)3-9, Enhancer-of-zeste, Trithorax; TTRF: Target to replication foci.

The Set1 H3K4 methyltransferases also appear to interact with unmethylated DNA, although in this case it is via an accessory protein, as was the case for Dnmt3a/Dnmt3L discussed earlier. Set1 lacks an intrinsic CXXC domain, but interacts directly with a protein that contains the same domain, Cfp1 [64,65]. High-throughput sequencing of Cfp1-bound chromatin identified a notable concordance between H3K4me3 and Cfp1 at unmethylated CpG islands in the mouse brain [66]. At loci that exhibit allele-specific DNA methylation (e.g., imprinted loci and the Xist gene), Cfp1 bound specifically to the unmethylated allele. Depletion of Cfp1 resulted in a marked genome-wide reduction in H3K4me3. The targeting of Cfp1 to CpG islands was independent of promoter activity, as the insertion of an unmethylated CpG-dense construct into the genome of embryonic stem cells was sufficient to nucleate Cfp1 binding and H3K4me3. This suggests that unmethylated CpGs recruit Cfp1 and the associated methyltransferase Set1 creates new marks of H3K4me3 on the local chromatin.

Like the histone H3K4 methyltransferases of the MLL/SET1 family, the Jumonji-domain containing histone demethylase JHDM1a (also known as CXXC8 or KDM2A) also has a CXXC domain (Figure 2A) [67]. Recent work indicates that, like Cfp1, JHDM1 is recruited to unmethylated CpG islands on a genome-wide scale, and that this is dependent on the CXXC domain [68]. The localization to CpG islands was independent of promoter activity and gene-expression levels, and correlated with the selective depletion of H3K36me1/2 within the CpG island but not surrounding regions or the bodies of genes; knockdown of KDM2A resulted in the selective accumulation of H3K36me2 in these regions. Consistent with a role for DNA methylation in restricting the localization of CXXC proteins, KDM2A was mislocalized to pericentric heterochromatin in Dnmt1^-/- mice. Furthermore, the lack of methylation alone does not appear to be sufficient for KDM2A recruitment in vivo as KDM2A does not localize to non-CpG island promoters that lack methylation. Although in vitro studies suggest that CXXC domains can bind a single CpG site with micromolar affinity, both the Cfp1 and KDM2A studies suggest that the targeting of CXXC proteins in vivo is dependent on CpG density as well as methylation status. It could be possible that these proteins oligomerize and form nucleoprotein filaments on CpG-dense DNA, in a manner similar to that described for Dnmt3a and -3L [69].

CXXC domains are also found in DNMT1, which is the enzyme responsible for generating 5-methyl-cytosine (5mC) [70], the MBD protein MBD1 (which binds 5mC) [71] and TET1, a Jumonji-like 2-oxoglutarate- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (Figure 2A) [72]. Interestingly, a recurrent (q22;q23) translocation [10,11] has been described in acute myelogenous leukemias, it results in a fusion transcript that juxtaposes the first six exons of MLL (containing an AT hook and CXXC) to the C-terminal third of TET1, thus ‘replacing’ the TET1 CXXC with the MLL CXXC [73,74]. Whether this leads to altered targeting of methyl hydroxylation remains to be determined.

Structurally, the CXXC domain has a novel fold in which two Zn ions are each coordinated tetrahedrally by four conserved cysteines; three from one CXXCXXC motif and one from a distal cysteine residue, resulting in an elongated shape (Figure 2B) [62]. The CXXC domain binds DNA in a clamp-like manner with the long axis of the structure linking the two Zn ions nearly perpendicular to the DNA axis (Figure 2B) [63]. The CpG-specific side-chain interactions involve a conserved (K/R/H)Q dipeptide (Figure 2B), except for the first and second CXXC domains of MBD1, which lack CpG-dependent DNA-binding activity [71]. A sequence alignment suggests that CXXC domains can be divided into two subfamilies, one with a longer DNA-binding loop containing a Lys–Phe–Gly–Gly motif, and the other, which is a shorter DNA-binding loop between cysteine 6 and cysteine 7 (Figure 2B). In the NMR structure of the MLL1 CXXC domain in complex with DNA [63], the Lys–Phe–Gly–Gly motif is located away from the DNA-binding surface (Figure 2B), suggesting a non-DNA-interacting function for this subfamily, which includes CFP1, JHDM1, DNMT1 and the third CXXC domain of MBD1.

Role for MBDs in the linkage between histone modification & DNA methylation

The MBD is present in a family of proteins conserved throughout the eukaryotic lineage. This domain, in some but not all cases, confers the ability to bind methylated CpGs. Mammals have five well-characterized members of this family, each with unique biological characteristics (Figure 3A) [75]. Of particular interest are recent reports indicating that in zebrafish, MBD4 (containing both a N-terminal MBD and a C-terminal thymine glycosylase domain [76]) and the cytidine deaminase AID cooperate to demethylate DNA [77]. Consistent with a role in DNA demethylation, erasure of DNA methylation in primordial germ cells is less efficient in AID-deficient animals [78], and AID is required to demethylate pluripotency genes during reprogramming of the somatic genome in embryonic stem cell fusions [79]. It is noteworthy that phosphorylation of MBD4 enhances DNA demethylation [80].

Figure 3. Methyl-CpG-binding domain-containing proteins.

(A) Model of the reactions that regulate H3K9 methylation by SUV39H1 or SETDB1. Recognition of methylated CpGs by the MBD of MBD1 directs methylation of H3K9me3. Inset: MBD structures of MeCP2 and MBD1 in complex with methyl-CpG DNA, respectively. (B) Sequence alignment of known and putative MBD domains. BAZ2-A and -B are two related proteins containing a MBD, DDT, PHD-type zinc finger and bromodomain. DDT is predicted to be a DNA binding domain (∼60 residues in length). BAZ2A, also known as TTF-I interacting peptide 5 or TIP5; hWALp3, is a component of the chromatin remodeling complex NoRC, a complex that mediates silencing of a fraction of rDNA.

DDT: DNA-binding homeobox and different transcription factors; HDAC: Histone deacetylase; MBD: Methyl-CpG-binding domain; PDB: Protein database; PHD: Plant homeodomain; SET: Su(var)3-9, Enhancer-of-zeste, Trithorax.

A particularly interesting observation, for the purpose of this article, is the fact that MBD1 forms a stable complex with SETDB1 [81,82], a H3K9 trimethylation (H3K9me3) methyltransferase responsible for the silencing of endogenous retroviruses [28,83,84], as well as the SUV39H1/HP1 complex [85], a heterochromatin-specific H3K9me3 writer and reader. SETDB1 also contains an intrinsic putative MBD domain with two conserved DNA-interacting arginine residues (Figure 3B) known to make direct contact with DNA in the structures of the MBD domain from MBD1 [86] and MeCP2 [87]. It remains to be seen whether the putative MBD domain of SETDB1 is similarly able to selectively bind methylated DNA. The intrinsic or associated coupling of a DNA methylation ‘reader’ with H3K9me3 ‘writers’ implies an interdependent mechanism for the propagation or maintenance of these marks. However, whereas DNA methylation is dependent on H3K9me3 in some organisms (the Arabidopsis and Neurospora examples noted earlier), it is independent of DNA methylation in others (mammals). Indeed, as noted above, mouse cells deficient in the SUV39H1 or SETDB1 H3K9 trimethyltransferases have relatively minor defects in DNA methylation, at least at constitutive heterochromatin or endogenous retroelements [27,28]. Likewise, although genetic or pharmacologic ablation of DNA methylation in mouse or human cells leads to a concomitant depletion of H3K9 dimethylation, it has little impact on H3K9me3 [27,88,89]. Thus, the functional relationships between DNA methylation and H3K9 methylation are complex, context-dependent and are likely to be further complicated by differences in the way that trimethylation versus dimethylation at H3K9 are initiated and maintained (the latter being catalyzed by G9a and G9a-like histone methyltransferases; reviewed in [90]).

As MBD1 contains two DNA-binding domains – an MBD domain (Figure 3) that recognizes methylated CpG, and a CXXC domain (Figure 2) that binds unmethylated CpG – it is unclear whether these two domains function independently to facilitate the recruitment of MBD1 to repressive complexes or active complexes, or if they cooperate in some way. Recently, Clouaire et al. found that the MBD domain of MBD1 binds more efficiently to methylated DNA within a specific-sequence context and that a functional MBD domain is necessary and sufficient for recruitment of MBD1 to these loci, while DNA binding by the CXXC domain is largely dispensable [91].

UHRF1 links hemi-methylated CpGs to histone modifications during replication

Hemi-methylated CpG sites, where only one DNA strand is methylated, are transiently generated during semi-conservative DNA replication. Methylation patterns are faithfully preserved with somatic cell division by a maintenance methylation reaction, involving both Dnmt1 and its accessory protein, UHRF1 [42,43], which maintains the fidelity of DNA methylation by recognizing these sites and selectively modifying the daughter strand CpG.

UHRF1 harbors five recognizable functional domains (Figure 1B): a ubiquitin-like domain at the N-terminus, followed by a tandem Tudor domain that binds H3K9me2/3 [92,93], an ADD-like domain that binds the histone H3 tail [46,47], a SET- and RING-associated (SRA) domain that binds hemi-methylated CpG-containing DNA [42,43,94–96] and a RING domain at the C-terminus that may endow UHRF1 with E3 ubiquitin ligase activity to histones [46]. As discussed earlier, Dnmt1 contains a CXXC domain (Figure 2) that binds unmethylated CpGs [70]. The interaction with UHRF1 provides a SRA domain that recognizes hemi-methylated CpGs, the substrate of Dnmt1. It is tempting to speculate that these two DNA-binding domains function synergistically to maintain the fidelity of DNA methylation during replication by preventing Dnmt1 from modifying unmethylated CpGs (through the masking of such sites by the CXXC domain) and directing methylation to newly synthesized hemi-methylated CpG sites (via the SRA domain). Indeed, molecular modeling suggests that the Dnmt1 catalytic domain and UHRF1 SRA domain are very unlikely to bind simultaneously to the same hemi-methylated CpG site [95]. Somehow the SRA domain must be displaced from the site to allow methylation by Dnmt1 (Figure 4).

Figure 4. Putative model of the coupling of UHRF1 and Dnmt1 to maintain the fidelity of DNA methylation during DNA replication.

The binding of the SRA domain of UHRF1 to hemi-methylated CpG sites generated by new strand synthesis recruits Dnmt1, allowing for displacement and subsequent methylation of the daughter strand.

SRA: SET and RING associated.

We therefore suggest that the SRA–DNA interaction (through recognition of and flipping of the parent strand 5mC out of the helix [94–96]) serves as an anchor to keep UHRF1 at the hemi-methylated CpG site where it then recruits Dnmt1 to methylate the opposing CpG in the daughter strand. The displacement of UHRF1 from the Dnmt1-substrate site is very similar to recently discovered components of the nucleotide excision repair pathway. Alkyltransferase-like proteins (ATL) share amino acid sequence homology with O₆-alkylguanine DNA alkyltransferases, but lack the active-site cysteine and alkyltransferase activity. Structures of Schizosaccharomyces pombe ATL bound to damaged DNA containing O₆-methylguanine or cigarette-smoke-derived oxobutylguanine demonstrate that the damaged nucleotide is flipped out of the DNA helix into a binding pocket [97]. However, the binding of ATL to the damaged strand inhibits repair by O₆-alkylguaninetransferase in vitro. Thus, ATL binding to the alkylated DNA base generates a stable complex that recruits the nucleotide excision repair enzymatic protein(s), which must have the ability to displace ATL from the lesion [97].

The identification of UHRF1 and its potential role in modulating the specificity of Dnmt1 for hemi-methylated CpG sites provides another layer to the mechanisms ensuring the faithful transmission of epigenetic information during DNA replication. Given that UHRF1 has the potential to interact with both hemi-methylated CpGs (via the SRA domain) and H3K9me2/3 (via the Tudor domain), and is known to interact with a wide variety of epigenetic regulators, including Dnmt1 [42,43], the H3K9 di- and mono-methyltransferase G9a [98] and a histone acetyltransferase Tip60 [99], it is possible that UHRF1 and the proteins in this larger complex play a more central role in coupling the transmission of DNA and histone methylation (H3K9 in particular) during mitotic cell division.

Future perspective

While it is well accepted that DNA methylation patterns are replicated in a semi-conservative fashion during cell division via the mechanisms discussed earlier, one of the fundamental unresolved questions is how, and indeed whether, histone modifications are similarly ‘inherited’. Recent work has shed light on this question. Through a combination of kinetic isotope labeling and mass spectroscopy studies, Xu et al. showed that histone H3.1–H4 tetramers remain intact and are partitioned evenly during DNA replication-dependent chromatin assembly [100]. Considering that the well-studied lysine methylation events reside on histone H3 (K4, K9, K27, K36 and K79) or H4 (K20), this evokes a model in which histone methylation patterns may be copied onto newly deposited tetramers from neighboring parental nucleosomes. Indeed, many of the SET domain histone methyltransferases contain intrinsic or associated reader domains that recognize the same mark that they generate, allowing for the copying of these marks from old to new nucleosomes. For example, G9a/GLP catalyzes H3K9me1/2 and contains an ankyrin-repeat domain that binds H3K9me1/2 [101]. Likewise, SUV39H1/2, the H3K9me3 writer interacts with HP1, the H3K9me3 reader [102]. Similarly, yeast Clr4 methylates H3K9 and contains a chromodomain that binds H3K9me3 [103]. Thus, higher organisms have evolved coordinated mechanisms of deposition and transmission of repressive chromatin marks to both DNA and histones.

In summary, epigenetic modifiers are often modular, containing distinct catalytic and regulatory domains. Many act as components of large complexes such that a complete understanding of their function will require the analysis of modular catalytic activity within the context of histones, DNA and protein partners. The web of interactions detailed in this article will likely benefit from a systems approach, such as those applied to signaling networks [104].