Methylation of DNA

Open Posted By: surajrudrajnv33 Date: 13/10/2020 Graduate Case Study Writing

Answer the questions below in an APA-style paper that is 1-3 pages in length (about 750 to 1000 words) about the Methylation of DNA

1. Give a basic description of the process. Please include at least one figure/diagram/flowchart of your own design.

2. What is the purpose of the process? What function/benefit can it provide?

4. Are there any key individuals identified in the original research that lead to the current accepted theories/model describing this process?

5. Choose three research articles (from after 2017) that focus on the process. Explain the importance of results/analysis in each paper that led to a greater understanding of this process. What important fact(s)/idea(s) did the paper/research reveal?

6. List all of your references in a Bibliography, using proper in-text citations throughout your answer.

Category: Mathematics & Physics Subjects: Algebra Deadline: 12 Hours Budget: $120 - $180 Pages: 2-3 Pages (Short Assignment)

Attachment 1

Edwards et al. Epigenetics & Chromatin (2017) 10:23 DOI 10.1186/s13072-017-0130-8


DNA methylation and DNA methyltransferases John R. Edwards1, Olya Yarychkivska2, Mathieu Boulard2 and Timothy H. Bestor2*

Abstract The prevailing views as to the form, function, and regulation of genomic methylation patterns have their origin many years in the past, at a time when the structure of the mammalian genome was only dimly perceived, when the num- ber of protein-encoding mammalian genes was believed to be at least five times greater than the actual number, and when it was not understood that only ~10% of the genome is under selective pressure and likely to have biological function. We use more recent findings from genome biology and whole-genome methylation profiling to provide a reappraisal of the shape of genomic methylation patterns and the nature of the changes that they undergo during gametogenesis and early development. We observe that the sequences that undergo deep changes in methylation status during early development are largely sequences without regulatory function. We also discuss recent findings that begin to explain the remarkable fidelity of maintenance methylation. Rather than a general overview of DNA methylation in mammals (which has been the subject of many reviews), we present a new analysis of the distribu- tion of methylated CpG dinucleotides across the multiple sequence compartments that make up the mammalian genome, and we offer an updated interpretation of the nature of the changes in methylation patterns that occur in germ cells and early embryos. We discuss the cues that might designate specific sequences for demethylation or de novo methylation during development, and we summarize recent findings on mechanisms that maintain methyla- tion patterns in mammalian genomes. We also describe the several human disorders, each very different from the other, that are caused by mutations in DNA methyltransferase genes.

Keywords: Epigenetics, DNA cytosine methylation, Mammalian DNA methyltransferases, Methylation dynamics, Methylation-related human diseases

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

The shape of genomic methylation patterns The current human genome assembly contains ~3 × 107 CpG dinucleotides, each of which can exist in the methyl- ated or unmethylated state. The number of possible meth- ylation patterns in a single haploid genome far exceeds the number of atoms in the observable universe; this greatly increases both the potential information content of the genome and the difficulty of statistical analysis [1, 2].

Whole-genome methylation profiling has recently made it possible to assign approximate methylation lev- els to the multiple sequence compartments that make up the human genome [1–3]. We have analyzed this

compartment-specific methylation as it occurs in dif- ferentiated somatic cells; the data are shown in Fig.  1. Transposon-derived sequences (SINE, LINE, and LTR) are abundant and densely methylated; the remainder of the genome is more variably methylated, with promoter- associated CpG islands and first exons representing the only sequence compartment that is largely unmethylated. Seventy-five percent of all promoters are within CpG islands and unmethylated [1–4]; the remaining promot- ers have very low CpG densities, and methylation is very unlikely to regulate their expression [1, 2]. Many CpG islands are not associated with promoters or other anno- tated regulatory sequences, and their methylation status is of unknown and possibly inconsequential  biological significance.

Current evidence indicates that the primary biologi- cal functions of DNA methylation lie in the heritable

Open Access

Epigenetics & Chromatin

*Correspondence: [email protected] 2 Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, NY, USA Full list of author information is available at the end of the article

Page 2 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

transcriptional repression of retrotransposons, the mon- oallelic expression of imprinted genes, X chromosome inactivation in female cells, and the selective exposure of promoters of cellular genes to transcription factors. There is evidence that genomic methylation patterns at regula- tory sequences are essentially static during development, although CpG-poor promoters can show partial demeth- ylation upon transcriptional activation that is likely to be a consequence rather than a cause of activation [2].

Multiple lines of evidence indicate that only ~10% of the mammalian genome is functional, as shown by comparative biology studies and by the fact that most of the genome is evolving at the neutral rate and does not appear to be under selection [6, 7]. Most DNA meth- ylation is also likely to be without significant biological function; this is consistent with high rate of loss of CpG dinucleotides across most of the genome during evolu- tion [8] and the highly heterogeneous nature of genomic methylation patterns even in single tissue types [1, 3].

Methylation dynamics during development Since 1987, it has been held that there are two waves of demethylation and remethylation [9] during develop- ment that in the standard depictions are implied to affect virtually the entire genome (reviewed in [10]). Under this

model, the first wave of demethylation occurs during the migration of proliferating primordial germ cells, with remethylation occurring in postmigratory germ cells; the second wave of demethylation takes place in cleav- age stage embryos and results in a minimum in DNA methylation at the blastocyst stage. As shown in Fig.  2, this standard double-dip model obscures the methyla- tion dynamics of the small fraction of the genome shown in Fig.  1 where methylation is likely to exert regulatory effects. First, the large majority of CpG island promot- ers are not subject to these waves of methylation and demethylation because they are unmethylated at all stages. Second, the methylation status of alleles at dif- ferentially methylated regions (DMRs) of imprinting control regions (ICRs) changes at different developmen- tal stages: they are demethylated in primordial germ cells and remethylated in cohorts of growing oocytes shortly before ovulation [11] and in the entire popula- tion of prospermatogonia around the time of birth [12]. The sex-specific methylation at ICRs/DMRs escapes the demethylation that occurs in cleavage stage embryos. Third, the small population of young, CpG-rich trans- posons largely escapes demethylation both in primordial germ cells [13] and in the early embryo [14]. The types of sequences that undergo the double wave of demeth- ylation and remethylation are largely composed of old and inactive transposon remnants, satellite and other repeated DNA, and the unannotated and rapidly diverg- ing fraction of the genome that shows little evidence of biological function. Figure 2 shows that the dynamics of demethylation and remethylation during development are more complex than depicted in the double-dip model and that sequences whose methylation status is of biolog- ical importance do not conform to this model.

Figure 2 also shows the basis for the pronounced sex- ual dimorphism in the rate of C → T mutations driven by deamination of 5 methylcytosine (m5C), which con- verts the base directly to T. De novo methylation of DMRs/ICRs and most of the genome occurs in the entire population of male germ cells around the time of birth; these methylation patterns exist for the repro- ductive life of the organism and must be propagated by maintenance methylation in spermatogonia through many mitotic divisions prior to entry into meiosis [12]. In female germ cells, de novo methylation takes place in growing oocytes, which are arrested in meiosis I and undergo no mitotic divisions prior to fertilization [11]; there is therefore very little opportunity for deamination of m5C to occur. As a result of this sexual dimorphism, de novo mutations at CpG dinucleotides are much more common in spermatozoa [15]; many sporadic genetic disorders are caused primarily by C →  T mutations at methylated CpG dinucleotides at alleles of paternal

Fig. 1 Distribution of DNA methylation across sequence compart- ments in the human genome. Vertical axis indicates percentage of total CpG dinucleotides in each indicated compartment; horizontal axis indicates percentage of total genome in each compartment; light blue at the top of each compartment indicates unmethylated fraction. Numerals in red denote CpG dinucleotides per 100 bp. The genome- wide CpG density expected on the basis of G + C content is 4.2 per 100 bp. Note that the only sequence compartment that exists in the largely unmethylated state is the CpG island/first exon compartment; this compartment occupies <0.5% of the genome. The ICR/DMR compartment (differentially methylated regions of imprinting control regions) represents ~0.001% of the genome and ~0.01% of total CpG dinucleotides. Introns are included in the unannotated compartment, as are putative enhancers. The methylation data are from Bisulfite-Seq data for hippocampus (Roadmap Epigenome Project sample E071 [5]), but other differentiated adult tissues show very similar trends

Page 3 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

origin. Furthermore, paternally methylated ICRs/DMRs have been eroded by C → T mutations over evolution- ary time and are far fewer in number and have become reduced in CpG density as compared to maternally methylated ICRs/DMRs [16].

Attracting and repelling DNA methylation The cues that designate specific sequences for de novo methylation, faithful versus error-prone maintenance methylation, or demethylation at different develop- mental stages are not well understood. It is clear that the default state of most of the genome is partially to

densely methylated [1, 3]. This is shown by the fact that removal of most DNA methylation in somatic cells by treatment with DNA methyltransferase inhibitors is fol- lowed by gradual remethylation of most sequences after withdrawal of the inhibitor [17]; this methylation occurs largely at sequences that are unlikely to have appreciable biological effects. Restoration of DNMT1 to Dnmt1-null ES cells, whose genomes have lost nearly all m5C, also results in the remethylation of most of the genome, but with a failure to reestablish methylation at imprinting control regions until these sequences have been passed through the germ line [18].

Fig. 2 Dynamics of demethylation and de novo methylation in the maternal (a) and paternal (b) genomes during mammalian development. The standard depictions of developmental changes in genomic methylation patterns often assume a monolithic genome; in fact, different sequence compartments display marked differences in timing of methylation and demethylation. CpG-rich (CpG island) promoters are unmethylated at all stages, except for the small number of CpG islands associated with imprinting control regions and CpG islands on the inactive X chromosome in somatic cells of females. Young, CpG-rich transposons largely escape both waves of demethylation. Most of the dynamic methylation and demethylation that occurs in primordial germ cells (PGCs) and the early embryo affects sequences that are evolving at the neutral rate and whose methylation status is without known biological effect. The methylation status of these sequences, which represent the bulk of the genome and are composed of satellite DNA, old and inactive transposons, introns, and unannotated sequences evolving at the neutral rate, is shown by broken lines

Page 4 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

Repeated sequences can attract de novo methylation; a transgene array of tandem repeats became methyl- ated in transgenic mice, but methylation was lost when the repeat array was reduced to a single unit [19]. Other mechanisms by which repeated sequences might be targeted for de novo methylation have been discussed [20], although the actual mechanism by which repeated sequences attract de novo methylation has not been defined.

The mechanisms that designate specific sequences for de novo methylation in the germ line are only partially understood. Deletion of the gene that encodes DNMT3L (which is related to DNMT3A and DNMT3B in frame- work regions but lacks the domains involved in trans- methylation) causes a failure of de novo methylation in prospermatogonia [12] and in growing oocytes [11], the only cell types in which DNMT3L is expressed. DNMT3L forms a complex with DNMT3A and DNMT3B, and DNMT3L targets this complex to DNA sequences asso- ciated with histones that are unmethylated at lysine 4 of histone H3 (H3K4); unmethylated H3K4 is associated with inactive promoters and with methylated DNA [21]. Ablation of the Argonaute proteins MILI or MIWI2, which are expressed in early germ cells and are involved in the biogenesis of PIWI-interacting RNAs (piRNAs), causes a failure of de novo methylation very similar to that seen in Dnmt3L-null germ cells, although DNA methylation is affected only in male germ cells [22]. This finding implies that piRNAs are upstream of histone H3K4 methylation and demethylation, which in turn are upstream of the DNMT3L/DNMT3A/DNMT3B com- plex. However, it is not known how piRNAs affect H3K4 methylation and no connection between piRNAs and the DNMT3L/DNMT3A/DNMT3B complex has been iden- tified. DNMT3A and DNMT3B have also been shown to bind to H3K36me3 through their PWWP domains [23].

The binding of transcription factors to promoters even in the absence of active transcription can cause the loss of DNA methylation in the vicinity of the bind- ing site [24]; even the binding of lac repressor can cause the loss of DNA methylation from CpG sites near lac operators in transfected mammalian cells [25]. Many of the expression–methylation correlations that have been reported since 1978 [26] are likely to be a consequence of transcriptional activation rather than a cause [2]. These effects are largely restricted to sequences of low CpG density. The expression of the large majority of genes does not markedly change after global genome demeth- ylation [2].

CpG island promoters are protected from de novo methylation at essentially all developmental stages. Exceptions are a small number of promoters at ICR/ DMRs [27] and CpG island promoters on the inactive

X chromosome in female somatic cells [28]. The mech- anism that protects CpG island promoters does not involve sequestration of the promoters in condensed chromatin since unmethylated CpG-rich sequences in nuclei show the greatest accessibility to diffusible factors such as DNase I [1].

Although the mechanisms that protect most CpG island promoters from de novo methylation are not understood, a specific class of CpG island promoters is protected from de novo methylation by the multidomain chromosomal protein FBXL10 (also known as KDM2B, JHDM1B, and CXXC2); these are the CpG island pro- moters bound by polycomb repressive complexes (PRC) 1 and 2. In the absence of FBXL10, PRC-bound pro- moters undergo de novo methylation with concomitant silencing of gene expression [29]. Even though FBXL10 is bound to essentially all CpG island promoters, removal of FBXL10 induces de novo methylation and transcrip- tional silencing only of that small subset of CpG island promoters that are bound both by FBXL10 and by PRC 1 and 2. This implies that PRC 1 and/or 2 has a tendency to attract de novo methylation and that FBXL10 has evolved to counteract this activity. Inhibition of de novo methyla- tion is likely to involve the CXXC domain of FBXL10; this domain, which is found in ~14 nuclear proteins, binds specifically to unmethylated CpG dinucleotides [30]. The methylation abnormalities that arise in cells that lack FBXL10 are strikingly similar to those seen in pediatric ependymomas and some other pediatric cancers; the methylation abnormalities appear to be important driv- ers of tumorigenesis as very few mutations have been detected in these tumors [31]. The processes that render PRC-bound promoters subject to de novo methylation in these tumors are not currently understood.

Mechanisms that mediate faithful maintenance methylation by DNMT1 That genomic methylation patterns are subject to mitotic inheritance in somatic cells was predicted to occur in 1975 [32, 33] and demonstrated experimentally in 1981 [34]. Maintenance methylation can be very faithful; allele-specific methylation patterns established at ICRs/ DMRs in germ cells of the preceding generation can be maintained in offspring through to adulthood with little alteration, and the two X chromosomes in female cells maintain different methylation patterns from soon after implantation of the embryo to the end of life.

However, maintenance methylation is less efficient at other sequences; methylation patterns can be heteroge- neous in single tissue types [1, 3] and even in clonal cell populations. Heterogeneous methylation is observed largely at sequences without discernable regulatory activity. DNMT1 has long been known to preferentially

Page 5 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

methylate hemimethylated DNA (the product of semi- conservative DNA replication; reviewed in [35]), but only recently have structural studies begun to reveal the mechanism.

As shown in Fig.  3, DNMT1 has a C-terminal cata- lytic domain related in sequence and structure to other DNA (cytosine-5) methyltransferases (including bacte- rial restriction methyltransferases such as M.HhaI) and a large N-terminal region that contains multiple functional domains. The CXXC domain (which is closely related to that of FBXL10) binds to unmethylated CpG dinu- cleotides and interposes a stretch of highly acidic amino acids (the autoinhibitory BAH1-CXXC linker in Fig.  3c and d) between the DNA and the active site of DNMT1, thereby inhibiting de novo methylation [30]. This auto- inhibitory mechanism provides a several-fold preference of DNMT1 for hemimethylated DNA, but this is not sufficient to explain the faithfulness of in  vivo mainte- nance methylation. A second mechanism that increases the preference of DNMT1 for hemimethylated DNA involves the interaction of the replication focus target- ing sequence (RFTS) of DNMT1 with the multidomain protein UHRF1 (ubiquitin-like with PHD and ring finger domains 1), which contains an SRA domain that binds to hemimethylated CpG dinucleotides [36]. In the free protein, the RFTS of DNMT1 occludes access of DNA to the active site by impingement on the CXXC domain; it is proposed that the UHRF1/hemimethylated DNA com- plex displaces the inhibitory RFTS domain of DNMT1 in a handoff reaction to transfer hemimethylated DNA from UHRF1 to the active site of DNMT1. This proposal is consistent with the finding that UHRF1 is required for maintenance methylation in  vivo; null alleles of Uhrf1 phenocopy null alleles of Dnmt1 in mice [36]. UHRF1 has multiple additional functional domains (including a tandem tudor domain that has been reported to bind to methylated H3K9 and to regulate the fidelity of mainte- nance methylation [37]), but mutation of UHRF1 so as to eliminate H3K9 binding had little effect on maintenance methylation in vivo [38].

DNMT1 also contains two bromo-adjacent homol- ogy (BAH) domains that occur in a number of other proteins, where some have been shown to bind to spe- cific modified histones (reviewed in [39]). The function of the BAH domains in DNMT1 is unknown, although they may increase the efficiency of maintenance meth- ylation by interaction with unidentified histones or his- tone modifications. While purified DNMT1 does have a modest intrinsic preference for hemimethylated DNA in vitro, it has recently become clear that multiple addi- tional regulatory inputs, especially those mediated by the interaction with UHRF1, are required in  vivo to ensure stable maintenance methylation at ICR/DMRs and other

sequences where DNA methylation is subject to stable somatic inheritance.

Pathogenic mutations in DNA methyltransferase genes All three human genes that encode active DNA methyl- transferases have been found to be mutated in specific human diseases, although gross methylation abnormali- ties have been observed only in ICF syndrome type 1 (immunodeficiency, centromere instability, facial anom- alies; OMIM 602900), which is caused by homozygous loss-of-function mutations at DNMT3B ([43]; Fig.  4a). ICF syndrome type 1 patients present with a variable combined immunodeficiency that is usually fatal prior to adulthood, mild but stereotypical facial abnormalities, and severe instability of classical satellite DNA on chro- mosomes 1, 9, and 16 that leads to gains and losses of chromosome arms to produce multiradiate or pinwheel chromosomes in phytohaemagglutinin (PHA)-stimu- lated T cells. Classical satellite DNA is almost completely unmethylated in all cells of patients with this syndrome, but chromosome instability is apparent only in cer- tain cell types. Variable losses of methylation in other regions of the genome have also been reported [44], but the methylation abnormalities responsible for the patho- genesis of ICF syndrome cannot be specified with any confidence. While point mutations in DNMT3B in ICF syndrome type 1 can eliminate all enzyme activity [43], ICF patients homozygous for deletion or early truncation alleles have not been reported. Null alleles of Dnmt3B in mice are embryonic lethals [45], which suggests that DNMT3B protein, even if enzymatically inactive, is required for proper assembly and function of a complex that contains other factors.

Heterozygous somatic mutations in DNMT3A are present in ~15% of cases of acute myeloid leukemia (AML; OMIM 601626) [46] and in a smaller percent- age of cases of myelodysplastic syndrome. Most muta- tions affect a single codon: R882 (encoded by CGC). C  →  T mutations at a methylated CpG dinucleotide within this codon convert it to a cysteine codon (TGC) if the top strand is mutated and to a histidine codon (CAC) if the bottom strand is mutated (Fig. 4b). It is not known whether methylation abnormalities are involved in those AML cases that bear mutations in DNMT3A. Mutations in DNMT3A are uncommon in neoplastic conditions other than certain leukemias and myelodys- plastic syndrome.

Tatton–Brown–Rahman syndrome (OMIM 602769) is an overgrowth syndrome that involves tall stature, char- acteristic facial anomalies, and variable intellectual dis- abilities. The original authors referred to this condition as DNMT3A overgrowth syndrome [47]. Patients are

Page 6 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

Fig. 3 Structure and regulation of DNMT1. a Functional domains in DNMT1. A nuclear localization sequence (NLS) and replication focus targeting sequence (RFTS) are closest to the N-terminus. A CXXC domain binds selectively to unmethylated CpG dinucleotides; this binding event interposes an acidic autoinhibitory loop between the active site and unmethylated DNA to inhibit de novo methylation [30]. The bromo-adjacent homol- ogy (BAH) domains 1 and 2 are of unknown function but are related in structure to BAH domains in other proteins that bind to specific modified histones (reviewed in [39]). A run of alternating lysine and glycine residues joins the multidomain N-terminal domain to the large C-terminal methyl- transferase domain, which is related in sequence and structure to all other DNA (cytosine-5) methyltransferases (reviewed in [35]). Letters below the diagram indicate the position of N-terminal truncations in the crystal structures shown in b–e. b Superposition of the structures of active DNMT1 [30] and M.HhaI, a bacterial restriction methyltransferase [40]. The methyltransferase domain of DNMT1 shows strong isostery with full-length M.HhaI. c Superposition of autoinhibited DNMT1 in complex with unmethylated DNA and active DNMT1 deleted for the CXXC and autoinhibitory loop domains in complex with hemimethylated DNA [41]. DNA can be seen to have accessed the catalytic pocket of DNMT1 in the active complex and to be very close to the S-adenosyl-l-homocysteine present in both complexes. d, e Impingement of the RFTS on the CXXC domain displaces the latter (curved arrow) into a conformation that inhibits binding of DNA [42]. It is proposed that the interaction of UHRF1 bound to hemimethyl- ated DNA causes a retraction of the RFTS domain to allow access of hemimethylated DNA to the active site of DNMT1 [42]

Page 7 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

heterozygous for germ line mutations in DNMT3A dif- ferent from those reported to occur somatically in AML (Fig.  4b). It is not known whether DNA methylation abnormalities are present in Tatton–Brown–Rahman

syndrome; the early-onset overgrowth phenotype is not inconsistent with defects in imprinted gene expres- sion. Although there are no methylation data on this point, there are strong phenotypic similarities among

Fig. 4 Each of the three DNMT genes is mutated in specific and diverse human syndromes. a DNMT3B bears recessive loss-of-function mutations in ICF syndrome type 1. b DNMT3A is mutated in dominant DNMT3A overgrowth syndrome and in subset of cases of acute myeloid leukemia and myelodysplastic syndrome. While most AML/MDS mutations affect codon 882, mutations at other positions also occur. c The RFTS domain of DNMT1 is subject to many different dominant mutations in a variable adult-onset cerebellar ataxia, deafness, dementia, and narcolepsy syndrome. The RFTS mediates interactions with replication foci during S phase (d) and with UHRF1. The positions of the amino acid substitutions within the structure of DNMT1 are shown in e. Only a subset of reported disease-associated mutations are shown for any of the three genes

Page 8 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

Tatton–Brown–Rahman syndrome, Weaver syndrome (associated with heterozygous missense mutations in EZH2; OMIM 277590), and Sotos syndrome (usually associated with heterozygous loss-of-function muta- tions in NSD1; OMIM 117550) and the imprinting disor- der Beckwith–Wiedemann syndrome (OMIM 130650), which strengthens the possibility that all these syndromes involve disruption of normal imprinted gene expression (reviewed in [47]).

Multiple dominant germ line mutations clustered in a single small domain of DNMT1 cause a heterogeneous group of adult-onset neurological disorders that include ataxia, sensorineural deafness, narcolepsy, dementia, psychosis, and other neurological and psychiatric abnor- malities (OMIM 126375 and 605712) that are collectively known as autosomal dominant DNMT1 complex disor- der [48]. All the known causative mutations involve sin- gle amino acid substitutions within the replication focus targeting sequence (RFTS; Fig.  4c), which mediates the interaction of DNMT1 and UHRF1 and the recruitment of DNMT1 into replication foci during S phase; in non-S phase cells, DNMT1 has a diffuse nucleoplasmic distri- bution (Fig. 4d). The locations of the causative mutations within the structure of DNMT1 are shown in Fig. 4e.

That mutations in DNMT1 should cause adult-onset neurological defects without involvement of other tissues is unexpected; partial loss-of-function alleles of Dnmt1 in mice cause pervasive developmental delays and high rates of leukemia without obvious neurological abnormalities [49]. Furthermore, adult neurons are postmitotic and perform little or no maintenance methylation. DNMT1 protein is nonetheless present at appreciable levels in neurons, and the mutated proteins show a tendency to form cytoplasmic aggregates when overexpressed in cul- tured cells [48]. It is likely that the toxicity of these aggre- gates (if they form in neurons of affected individuals), rather than an effect on DNA methylation, underlies the neuropathies caused by mutations in DNMT1. This inter- pretation is consistent with the report of a lack of obvious phenotypes after conditional deletion of the Dnmt1 gene in postmitotic neurons of mice [50].

DNMT3L, which is expressed only in prospermato- gonia and in growing oocytes, recruits DNMT3A and DNMT3B to nucleosomes that contain unmethylated H3K4 [51]. No disease-associated mutations in the DNMT3L gene have been reported in humans; based on mouse models, homozygous null alleles would be expected to produce non-syndromic azoospermia in males and maternal-effect embryonic lethality in the off- spring of homozygous mutant females and normal males [11, 12].

The human biology of DNA methyltransferases illus- trates the complex and enigmatic effects of disturbances

of genomic methylation patterns on phenotype. The four human disorders firmly associated with mutations in DNA methyltransferase genes have largely non-overlap- ping phenotypes: one is germ line, recessive, early onset and involves a usually severe combined immunodefi- ciency (ICF syndrome type 1), one is germ line, domi- nant, adult onset and progressive and affects the central nervous system (DNMT1 complex disorder), another is germ line, dominant, early onset and involves overgrowth and intellectual disabilities without pronounced neuro- logical disturbance (Tatton–Brown–Rahman or domi- nant DNMT3A overgrowth syndrome), and another is somatic, dominant and is involved in the etiology of lym- phoid neoplasms (DNMT3A mutations at codon R882). Methylation abnormalities are likely to be involved in the causation of all the conditions. Given the vast num- ber of methylation patterns that can exist on a single genome and the high likelihood that the DNA methyl- transferase mutations will cause genome-wide meth- ylation abnormalities, it might be extremely difficult to identify the specific methylation change that gives rise to a given biological effect in the DNA methyltransferase disorders. A recent report of characteristic methylation anomalies in DNMT3AR882H/+ or DNMT3AR882C/+ cases of AML that are not present in DNMT3A+/+ cases [52] highlights the issue: while the data strongly indicate that abnormal genomic methylation patterns are involved in the progression to AML, the methylation changes actu- ally directly involved in leukemogenesis will be difficult to define.

Conclusions Many of the accepted views of the form, function, and dynamics of mammalian genomic methylation patterns were first formulated in the 1980s, when there was little information as to the true organization of the genome. A reappraisal in view of modern information leads to the conclusion that the dynamic demethylation and remethylation that occurs in early development affect largely unannotated sequences and inactive transposons, while imprinting control regions and potentially active transposons largely escape demethylation, and nearly all CpG-rich promoters are not methylated in any cell type. Genomic methylation patterns at sequences where methylation status might affect phenotype are much more static than previously believed, while methylation changes at sequences that are evolving at close to the neutral rate are unlikely to have biological consequences.

Abbreviations BAH domain: bromo-adjacent homology domain; CXXC domain: protein fold found in multiple proteins that bind to unmethylated CpG dinucleotides in DNA; DNMT: DNA (cytosine-5) methyltransferase; PRC1 and 2: polycomb

Page 9 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

repressive complexes 1 and 2. Involved in transcriptional repression, especially of genes involved in patterning the early embryo; RFTS domain: replication focus targeting sequence that recruits DNMT1 to replication foci during S phase.

Authors’ contributions JRE performed the computational analysis, OY conducted the structural analy- ses, and JRE, OY, MB, and THB wrote the paper. All authors read and approved the final manuscript.

Author details 1 Center for Pharmacogenomics, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA. 2 Department of Genetics and Development, College of Physicians and Surgeons of Columbia University, New York, NY, USA.

Acknowledgements This study was supported by grants from the NIH to JRE and THB.

Competing interests The authors declare that they have no competing interests

Consent for publication All authors have approved the manuscript.

Funding This study was supported by grants from the NIH to JRE and THB.

Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.

Received: 21 February 2017 Accepted: 26 April 2017

References 1. Edwards JR, et al. Chromatin and sequence features that define the fine

and gross structure of genomic methylation patterns. Genome Res. 2010;7:972–80.

2. Bestor TH, Edwards JR, Boulard M. Notes on the role of dynamic DNA methylation in mammalian development. Proc Natl Acad Sci USA. 2015;112:6796–67999.

3. Lister R, et al. Human DNA methylomes at base resolution show wide- spread epigenomic differences. Nature. 2009;462:315–22.

4. Ioshikhes IP, Zhang MQ. Large-scale human promoter mapping using CpG islands. Nat Genet. 2000;26:61–3.

5. Roadmap Epigenomics Consortium, Kundaje A, Meuleman W, Ernst J, Bilenky M, Yen A, Heravi-Moussavi A, Kheradpour P, Zhang Z, Wang J, et al. Integrative analysis of 111 reference human epigenomes. Nature. 2015;518:317–30.

6. Eddy SR. The ENCODE project: missteps overshadowing a success. Curr Biol. 2013;2:R259–61.

7. Graur D, Zheng Y, Price N. Azevedo RB, Zufall RA, Elhaik E. On the immor- tality of television sets: “function” in the human genome according to the evolution-free gospel of ENCODE. Genome Biol Evol. 2013;5:578–590.

8. Louie E, Ott J, Majewski J. Nucleotide frequency variation across human genes. Genome Res. 2003;13:2594–601.

9. Monk M, Boubelik M, Lehnert S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development. 1987;99:371–82.

10. Smallwood SA, Kelsey G. De novo DNA methylation: a germ cell perspec- tive. Trends Genet. 2012;28:33–42.

11. Bourc’his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the estab- lishment of maternal genomic imprints. Science. 2001;294:2536–9.

12. Bourc’his D, Bestor TH. Meiotic catastrophe and retrotransposon reactiva- tion in male germ cells lacking Dnmt3L. Nature. 2004;431:96–9.

13. Seisenberger S, Andrews S, Krueger F, Arand J, Walter J, Santos F, Popp C, Thienpont B, Dean W, Reik W. The dynamics of genome-wide DNA

methylation reprogramming in mouse primordial germ cells. Mol Cell. 2012;48:849–62.

14. Lees-Murdock DJ, De Felici M, Walsh CP. Methylation dynamics of repetitive DNA elements in the mouse germ cell lineage. Genomics. 2003;82:230–7.

15. Mugal CF, Ellegren H. Substitution rate variation at human CpG sites correlates with non-CpG divergence, methylation level and GC content. Genome Biol. 2011;12:R58.

16. Bourc’his D, Bestor TH. Origins of extreme sexual dimorphism in genomic imprinting. Cytogenet Genome Res. 2006;113:36–40.

17. Flatau E, Gonzales FA, Michalowsky LA, Jones PA. DNA methylation in 5-aza-2’-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells. Mol Cell Biol. 1984;4:2098–102.

18. Tucker KL, et al. Germ-line passage is required for establishment of meth- ylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev. 1996;10:1008–20.

19. Garrick D, Fiering S, Martin DI, Whitelaw E. Repeat-induced gene silencing in mammals. Nat Genet. 1998;18:56–9.

20. Bestor TH, Tycko B. Creation of genomic methylation patterns. Nat Genet. 1996;12:363–7.

21. Ooi SK, et al. DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature. 2007;448:714–7.

22. Aravin AA, Sachidanandam R, Bourc’his D, Schaefer C, Pezic D, Toth KF, Bestor T, Hannon GJ. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol Cell. 2008;31:785–99.

23. Rondelet G, Dal Maso T, Willems L, Wouters J. Structural basis for recogni- tion of histone H3K36me3 nucleosome by human de novo DNA methyl- transferases 3A and 3B. J Struct Biol. 2016;194:357–67.

24. Matsuo K, Silke J, Georgiev O, Marti P, Giovannini N, Rungger D. An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA. EMBO J. 1998;17:1446–53.

25. Han L, Lin IG, Hsieh CL. Protein binding protects sites on stable episomes and in the chromosome from de novo methylation. Mol Cell Biol. 2001;21:3416–24.

26. Waalwijk C, Flavell RA. DNA methylation at a CCGG sequence in the large intron of the rabbit beta-globin gene: tissue-specific variations. Nucl Acids Res. 1978;5:4631–4.

27. Proudhon C, et al. Protection against de novo methylation is instrumental in maintaining parent-of-origin methylation inherited from the gametes. Mol Cell. 2012;47:909–20.

28. Cotton AM, Price EM, Jones MJ, Balaton BP, Kobor MS, Brown CJ. Land- scape of DNA methylation on the X chromosome reflects CpG density, functional chromatin state and X-chromosome inactivation. Hum Mol Genet. 2015;24:1528–39.

29. Boulard M, Edwards JR, Bestor TH. FBXL10 protects Polycomb-bound genes from hypermethylation. Nat Genet. 2015;47:479–85.

30. Song J, Rechkoblit O, Bestor TH, Patel DJ. Structure of DNMT1-DNA com- plex reveals a role for autoinhibition in maintenance DNA methylation. Science. 2011;331:1036–40.

31. Mack SC, et al. Epigenomic alterations define lethal CIMP-positive epend- ymomas of infancy. Nature. 2014;506:445–50.

32. Holliday R, Pugh JE. DNA modification mechanisms and gene activity during development. Science. 1975;187:226–32.

33. Riggs AD. X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet. 1975;14:9–25.

34. Wigler M, Levy D, Perucho M. The somatic replication of DNA methyla- tion. Cell. 1981;24:33–40.

35. Goll MG, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Rev Biochem. 2005;74:481–514.

36. Bostick M, Kim JK, Estève PO, Clark A, Pradhan S, Jacobsen SE. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science. 2007;317:1760–4.

37. Rothbart SB, et al. Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation. Nat Struct Mol Biol. 2012;19:1155–60.

38. Zhao Q, et al. Dissecting the precise role of H3K9 methylation in cross- talk with DNA maintenance methylation in mammals. Nat Commun. 2016;7:12464.

39. Yang N, Xu RM. Structure and function of the BAH domain in chromatin biology. Crit Rev Biochem Mol Biol. 2013;48:211–21.

40. Klimasauskas S, Kumar S, Roberts RJ, Cheng X. HhaI methyltransferase flips its target base out of the DNA helix. Cell. 1994;76:357–69.

Page 10 of 10Edwards et al. Epigenetics & Chromatin (2017) 10:23

• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research

Submit your manuscript at www.biomedcentral.com/submit

Submit your next manuscript to BioMed Central and we will help you at every step:

41. Song J, Teplova M, Ishibe-Murakami S, Patel DJ. Structure-based mecha- nistic insights into DNMT1-mediated maintenance DNA methylation. Science. 2012;335:709–12.

42. Takeshita K, Suetake I, Yamashita E, Suga M, Narita H, Nakagawa A, Tajima S. Structural insight into maintenance methylation by mouse DNA meth- yltransferase 1 (Dnmt1). Proc Natl Acad Sci USA. 2011;108:9055–9.

43. Xu G-L, Bestor TH, Bourc’his D, Hsieh CL, Tommerup N, Bugge M, Hulten M, Qu X, Russo JJ, Viegas-Péquignot E. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyl- transferase gene. Nature. 1999;402:187–91.

44. Simo-Riudalbas L, Diaz-Lagares A, Gatto S, Gagliardi M, Crujeiras AB, Mata- razzo MR, Esteller M, Sandoval J. Genome-wide DNA methylation analysis identifies novel hypomethylated non-pericentromeric genes with poten- tial clinical implications in ICF syndrome. PLoS ONE. 2015;10(7):e0132517.

45. Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian develop- ment. Cell. 1999;99:247–57.

46. Ley TJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363:2424–33.

47. Tatton-Brown K. NSD1, EZH2 and DNMT3A overgrowth genes and their associated overgrowth syndromes. Chichester: Wiley; 2014.

48. Baets J, et al. Defects of mutant DNMT1 are linked to a spectrum of neurological disorders. Brain. 2015;138:845–61.

49. Gaudet F, Rideout WM 3rd, Meissner A, Dausman J, Leonhardt H, Jaenisch R. Induction of tumors in mice by genomic hypomethylation. Science. 2003;300:489–92.

50. Fan G, et al. DNA hypomethylation perturbs the function and survival of CNS neurons in postnatal animals. J Neurosci. 2001;21:788–97.

51. Ooi SK, Qiu C, Bernstein E, Li K, Jia D, Yang Z, Erdjument-Bromage H, Tempst P, Lin SP, Allis CD, Cheng X, Bestor TH. DNMT3L connects unmeth- ylated lysine 4 of histone H3 to de novo methylation of DNA. Nature. 2007;448:714–7.

52. Spencer DH, Russler-Germain DA, Ketkar S, Helton NM, Lamprecht TL, Fulton RS, Fronick CC, O’Laughlin M, Heath SE, Shinawi M, Westervelt P, Payton JE, Wartman LD, Welch JS, Wilson RK, Walter MJ, Link DC, DiPersio JF, Ley TJ. CpG Island hypermethylation mediated by DNMT3A is a conse- quence of AML progression. Cell. 2017;168(5):801–16.

Reproduced with permission of copyright owner. Further reproduction prohibited without permission.

  • DNA methylation and DNA methyltransferases
    • Abstract
    • The shape of genomic methylation patterns
    • Methylation dynamics during development
    • Attracting and repelling DNA methylation
    • Mechanisms that mediate faithful maintenance methylation by DNMT1
    • Pathogenic mutations in DNA methyltransferase genes
    • Conclusions
    • Authors’ contributions
    • References

Attachment 2

genes G C A T




The Growing Complexity of UHRF1-Mediated Maintenance DNA Methylation

Si Xie and Chengmin Qian *

School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China; [email protected] * Correspondence: [email protected]; Tel.: +0852-39-176-820.

Received: 1 November 2018; Accepted: 29 November 2018; Published: 3 December 2018 ���������� �������

Abstract: Mammalian DNMT1 is mainly responsible for maintenance DNA methylation that is critical in maintaining stem cell pluripotency and controlling lineage specification during early embryonic development. A number of studies have demonstrated that DNMT1 is an auto-inhibited enzyme and its enzymatic activity is allosterically regulated by a number of interacting partners. UHRF1 has previously been reported to regulate DNMT1 in multiple ways, including control of substrate specificity and the proper genome targeting. In this review, we discuss the recent advances in our understanding of the regulation of DNMT1 enzymatic activity by UHRF1 and highlight a number of unresolved questions.

Keywords: DNA methylation; DNMT1; UHRF1; USP7; ubiquitination

1. Introduction

DNA methylation is one of the best-characterized epigenetic changes that have a critical role in numerous biological processes including gene expression, genomic imprinting, X chromosome inactivation and genome stability [1–3]. In mammals, DNA methylation predominantly occurs at the C5 position of cytosine in CpG dinucleotides [4,5]. DNA methylation is catalyzed by the DNA methyltransferase family members including DNMT1, DNMT3A and DNMT3B. Another enzymatically inactive member DNMT3L interacts with both DNMT3A and DNMT3B and stimulates their enzymatic activity.

Methylation on CpG sites in promoters generally leads to gene repression by either inhibiting transcription factor binding or recruiting the repressive complex to the promoter region [6]. Therefore, selective methylation on promoter regions of certain genes facilitates the establishment of specific gene expression pattern in differentiated cells. The methylation pattern is tissue-specific and built mainly by de novo DNA methyltransferases DNMT3A and DNMT3B [7–9]. Once established, maintenance DNA methyltransferase DNMT1 ensures that the methylation pattern is faithfully propagated throughout successive cell divisions to maintain cell-specific functions in differentiated cells. During the replication process, DNMT1 preferentially catalyzes the conversion of hemi-methylated CpG dinucleotides in daughter strands to fully methylated forms. It is worthy to note that DNMT3A and DNMT3B have been found to be involved in maintaining DNA methylation patterns in specific loci, whereas DNMT1 exhibits de novo DNA methylation under certain circumstances [10–12]. In addition, the DNA methylation pattern is dynamic during the developmental processes and to maintain the balance between DNA methylation and demethylation could have a great impact on human health and disease. Similar to other epigenetic modifications, DNA methylation is reversible. 5-Methyl cytosine (5mC) can be further converted to 5-hydroxymethyl cytosine (5hmC), 5-formyl cytosine (5fC) and 5-carboxyl cytosine (5caC) by the DNA methyl-cytosine dioxygenases TET1, TET2 and TET3. It should be pointed out that 5hmC, 5fC and 5caC not just serve as DNA demethylation intermediates but also have distinct regulatory functions in various developmental processes [13,14].

Genes 2018, 9, 600; doi:10.3390/genes9120600 www.mdpi.com/journal/genes

Genes 2018, 9, 600 2 of 12

Mounting evidence has demonstrated that DNMT1 alone is not sufficient to maintain the global DNA methylation throughout cell division. Indeed, structural and biochemical analyses have suggested that DNMT1 is a self-inhibited enzyme, as DNMT1 N-terminal regulatory domains including replication foci targeting sequence (RFTS) domain and CXXC domain show autoinhibitory effect on DNMT1 enzymatic activity. Therefore, additional protein factors are required to release DNMT1 from its self-inhibited state. UHRF1 has emerged in recent studies to be one of such regulators [15–17]. In this review, we summarize the most recent findings on UHRF1-mediated regulation of DNMT1 recruitment and activation.

2. DNMT1 is an Autoinhibited DNA Methyltransferase

DNMT1 is a multi-modular protein consisting of a DMAP1-binding domain, RFTS domain, a CXXC domain, two BAH domains and a C-terminal catalytic domain (Figure 1A). Numerous studies have shown that N-terminal domains of DNMT1 have key regulatory functions. For example, the DMAP1-binding domain and RFTS domain together with a PCNA-interacting protein (PIP) box inserted between them are mainly responsible for DNMT1 stability and its proper localization onto DNA replication site [18–20]. At the same time, DNMT1 RFTS domain and CXXC domain can regulate the activity of the catalytic domain. The CXXC domain specifically binds to unmethylated CpG sequences in DNA and positions the CXXC-BAH1 linker between DNA and the catalytic pocket to prevent de novo methylation on those CpG dinucleotides [21,22].

Figure 1. Structures of DNMT1 and UHRF1. (A) Domain architecture of DNMT1 and crystal structure of human DNMT1 (aa: 351-1600; PDB: 4WXX) [23]. (B) Domain organization of UHRF1 and crystal structures of mouse UHRF1 UBL domain (PDB: 2FAZ), TTD-PHD in complex with H3K9me3 peptide (PDB: 3ASK), SRA in complex with hemi-methylated DNA fragment (PDB: 3CLZ) and UHRF1 RING finger (PDB: 3FL2) [24,25].

Earlier studies have revealed that RFTS domain regulates DNMT1 enzymatic activity through an auto-inhibitory mechanism, as explained by structural analysis showing that RFTS domain occupies DNMT1 catalytic pocket and prevents substrate DNA binding (Figure 2) [23,26]. Consistent with the structural observation that RFTS domain has intramolecular interaction with the catalytic domain (CD), disruption of RFTS-CD interaction strengthens the RFTS binding to histone H3, which led

Genes 2018, 9, 600 3 of 12

to abnormal DNMT1 accumulation on chromatin during S-phase [27] and aberrant increase on DNMT1 activity both in vitro and in vivo [28,29]. Such fine-tuned regulatory mechanism of DNMT1 methyltransferase activity could guarantee the faithful inheritance of DNA methylation pattern and maintain tissue-specific functions throughout cell division. Therefore, RFTS has been proposed as a fail-safe lock which protects the genome from aberrant replication-independent DNA methylation [30].

Figure 2. The DNMT1 replication foci targeting sequence (RFTS) domain regulates DNMT1 enzymatic activity through an auto-inhibitory mechanism. (A) Crystal structure of human DNMT1 (aa: 351-1600; PDB: 4WXX) in free form [23]. (B) Crystal structure of mouse DNMT1 (aa:731-1602; PDB: 4DA4) bound by hemimethylated CpG DNA [31]. (C) Superimposition of structures in (A) and (B) revealed that the RFTS domain occupies DNMT1’s catalytic pocket and prevents substrate DNA binding.

3. UHRF1 is Required for Proper Loading of DNMT1 onto Chromatin

UHRF1 is a multifunctional epigenetic regulator that bridges DNA methylation with multiple histone post-translational modifications such as histone methylation, ubiquitination and acetylation. A number of studies have demonstrated that UHRF1 interacts directly with DNMT1 and is essential in DNA methylation maintenance (Figure 1B). The UHRF1 SRA domain specifically binds to hemi-methylated DNA [15,16,25,32–34], that helps direct DNMT1 to natural substrate sites. It has also been reported that UHRF1 SRA domain interacts with DNMT1 RFTS domain which could promote the access of DNMT1’s catalytic center to hemi-methylated DNA [29,35]. The UHRF1 PHD finger and tandem tudor domain (TTD) cooperatively recognize H3R2 and H3K9me2/3 mark that may facilitate the proper localization of DNMT1 on genomic loci [24,36–43]. The UHRF1 C-terminal RING finger domain has been shown to function as an E3 ubiquitin ligase responsible for histone H3 ubiquitination [44,45]. Ubiquitinated histone H3 is subsequently recognized by DNMT1 that promotes recruitment of DNMT1 to DNA replication sites [46,47].

4. Allosteric Regulation of UHRF1 E3 Ubiquitin Ligase Activity

UHRF1 is proposed to adopt a closed autoinhibited conformation due to extensive inter-domain interactions within the protein [48–50]. Getalo et al. reported that a polybasic region (PBR) present between SRA and RING finger of UHRF1 serves as a competitive inhibitor of H3K9me2/3 interaction with the UHRF1 TTD, while the binding of phosphatidylinostiol phosphate (PI5P) to PBR allosterically relieves the inhibition [48]. More recent studies have demonstrated that PBR is a versatile platform that interacts with other partners such as USP7 and DNMT1 [49,51,52]. UHRF1 TTD interacts directly with a histone H3K9-like mimic within DNA ligase 1 (LIG1) [53]. All these interactions can similarly trigger UHRF1 conformational changes and regulate intramolecular activation of UHRF1. On the other hand, the binding of hemi-methylated DNA also causes conformational changes in UHRF1 [49], potentially releases TTD and allosterically regulates UHRF1 E3 ubiquitin ligase activity [50]. Multivalent engagement of hemi-methylated linker DNA and H3K9me2 enhances the enzymatic activity of UHRF1 toward nucleosomal histone substrates [54].

Genes 2018, 9, 600 4 of 12

Moreover, UHRF1 UBL domain has just been reported to allosterically regulate UHRF1 E3 ligase activity towards histone H3 [55,56]. UHRF1 UBL and RING finger interact with the ubiquitin conjugating E2 enzyme UbcH5a to stimulate histone H3 ubiquitination.

5. UHRF1-Dependent Histone H3 Ubiquitination Stimulates DNMT1 Enzymatic Activity

Nishiyama et al. first demonstrated that UHRF1 can ubiquitinate histone H3 on K23, and ubiquitinated H3 is subsequently recognized by DNMT1 [46]. The process actually facilitates the recruitment of DNMT1 to the replication foci. Several studies have further shown that a number of lysine residues including K14, K18 and K23 on histone H3 can be ubiquitinated by UHRF1 [47,50]. Two recent structure-based studies have reported crystal structures of RFTS in complex with ubiquitin and ubiquitinated histone H3 (H3-K18Ub/K23Ub) respectively, which shed light on the molecular mechanism of how UHRF1-dependent H3 ubiquitination regulates DNA methylation maintenance [57,58]. Notably, the RFTS domain of DNMT1 binds ubiquitin with 1:2 stoichiometry in both structures (Figure 3A,B). Superimposition of two complex structures gives a RMSD of 0.78 Å (Figure 3C), consistent with almost the same RFTS/ubiquitin interaction network observed in both structures. Although the ubiquitin-conjugated histone H3 is directly involved in RFTS interaction, it has no obvious effect on ubiquitin recognition by RFTS. This is consistent with the notion in the field that ubiquitin-conjugated substrates usually have no visible impact on the interaction between ubiquitin and ubiquitin-binding domains. RFTS binds to two mono-ubiquitinated histone H3 (H3Ub2) approximately 100 times stronger than histone H3 alone. Intriguingly, RFTS exhibits similar binding affinities to single mono-ubiquitinated histone H3 (H3Ub) and the unmodified H3, suggesting that binding of RFTS to histone H3 and two covalently attached ubiquitin molecules is cooperative [57]. Consistently, mutations on RFTS residues that only participate in the interaction with one of the bound ubiquitin molecules, completely abolish the RFTS association with both ubiquitin molecules, implying that binding of two ubiquitin molecules is interdependent and single mono-ubiquitinated histone H3 is insufficient for DNMT1 recruitment [57,58]. More interestingly, in vitro DNA methylation assays also demonstrate that only two mono-ubiquitinated histone H3, but not ubiquitin alone or unmodified histone H3 could stimulate DNMT1 enzymatic activity [57,58]. However, currently, available structures are unable to explain how exactly the dual mono-ubiquitinated histone H3 alleviates the RFTS auto-inhibitory effect. Further efforts on structural analysis of the complex of two mono-ubiquitinated histone H3 with DNMT1 including RFTS and catalytic domain are required to demonstrate the detailed conformational changes in DNMT1 upon ubiquitinated histone H3 binding.

Figure 3. The DNMT1 RFTS domain is a dual mono-ubiquitinated histone H3 binding module. (A) Crystal structure of DNMT1 RFTS in complex with ubiquitinated histone H3 (PDB: 5WVO) [57]. (B) Crystal structure of DNMT1 RFTS in complex with ubiquitin (PDB: 5YDR) [58]. (C) The interaction between RFTS and ubiquitin is essentially the same in these structures. Superimposition of two complex structures gives an RMSD of 0.78Å.

Genes 2018, 9, 600 5 of 12

6. DNMT1 Recruitment and Stimulation by UHRF1 N-Terminal UBL Domain

Considering that UHRF1 N-terminal UBL domain structurally resembles ubiquitin, Li et al. went on to test if the UBL domain of UHRF1 could interact with DNMT1 RFTS domain [58]. Indeed, UHRF1 UBL was found to physically interact with DNMT1 RFTS domain. Strikingly, the interaction network of RFTS-UBL is distinct from that of RFTS-ubiquitin although UBL shares high structural similarity with ubiquitin. For example, mutations on RFTS residues that abolished ubiquitin binding showed little or no effect on the interaction with UBL. Unexpectedly, the authors also found that interaction between DNMT1 and UHRF1 UBL could stimulate DNMT1 enzymatic activity. UHRF1 UBL could stimulate the enzymatic activity of DNMT1 segments (aa: 621-1616) and (aa: 351-1616) to a similar level, but H3Ub2 shows no stimulatory effect toward DNMT1(aa: 621-1616), suggesting distinct stimulatory mechanisms by UHRF1 UBL domain and ubiquitinated histone H3 [58]. The authors were able to figure out that UHRF1 UBL not only physically interacts with RFTS but also binds to DNMT1 (aa: 621-1616) and the binding to this DNMT1 fragment is essential for its stimulatory effect on DNMT1 enzymatic activity. It is worthwhile elucidating the detailed molecular basis of the interaction between UHRF1 UBL and DNMT1 in future studies.

UHRF1 UBL has also been revealed to interact with the ubiquitin conjugating E2 enzyme UbcH5a to stabilize the active E2/UHRF1/ubiquitin complex and direct mono-ubiquitination towards histone H3 [55,56], which eventually facilitates the recruitment of DNMT1 to replication foci for efficient DNA methylation maintenance.

7. Epigenetic Crosstalk between DNA Methylation and Histone Modifications in Maintenance DNA Methylation

Mounting evidence suggests that crosstalk between DNA methylation and histone modifications occurs to maintain DNA methylation patterns during replication. Previous reports have indicated that binding of UHRF1 to H3K9me2/3 is indispensable for DNMT1 association with chromatin. Disruption of the interaction between UHRF1 and H3K9me2/3 or H3R2 leads to loss of DNMT1 chromatin localization and global hypomethylation [36,41,59]. In contrast, a recent study demonstrated that although disruption of UHRF1 and H3K9me2/3 association leads to hypomethylation, the reduction of DNA methylation occurs globally and does not restrict in the H3K9me2/3 enriched region, suggesting that DNMT1 mediating DNA maintenance methylation is largely independent of H3K9 methylation [59]. Actually, this observation is not surprising considering that the UHRF1 PHD finger but not TTD plays a dominant role in histone H3 binding [24,40,43,60], and expression of UHRF1 with a defective PHD finger failed to restore DNA methylation pattern in UHRF1 knockout cells [47]. The UHRF1 PHD finger recognizes the histone H3 N-terminal tail with unmodified R2. Consequently, methylation of H3R2 by PRMT6 impairs chromatin binding of UHRF1 and induces global DNA hypomethylation [61]. Interestingly, a recent report suggested that side chain guanidinium group of histone H3R8 forms one hydrogen bond with the carbonyl oxygen atom of DNMT1 Tyr564, and forms another hydrogen bond with the side chain of DNMT1 Glu572 [57]. Two previous studies showed that side chain NηH atoms of histone H3R8 form hydrogen bonds with the carbonyl oxygen atom of UHRF1 Asp190 [24,60]. It is likely that methylation on histone H3R8 will antagonize its binding to DNMT1 and UHRF1 based on the above three structure-based analyses.

Histone H3 N-terminal residues such as K14, K18 and K23 can be acetylated or ubiquitinated, acetylation of these residues would preclude ubiquitination on the same sites. Previous studies have shown that HDAC1/2 associate with nascent chromatin during replication [62], DNMT1 and HDAC1/2 reside in a large protein complex [19,63], the presence of HDACs likely makes H3K14, H3K18 and H3K23 sites accessible to UHRF1 for mono-ubiquitination at replicating chromatin of specific genomic loci. On the other hand, acetylation on these lysine residues occurs more frequently at transcriptionally active regions where DNA methylation is rare. Furthermore, a number of studies have shown that DNMT1 and UHRF1 are subject to acetylation, ubiquitination and other post-translational modifications. These post-translational modifications (PTMs) could have an impact on protein stability,

Genes 2018, 9, 600 6 of 12

modulating protein-protein interactions and even their enzymatic activities, adding another layer of complexity to the regulatory mechanism of maintenance DNA methylation. Future studies are needed to examine if these epigenetic marks are sequentially erased or established during maintenance DNA methylation.

8. A Working Model for DNMT1/UHRF1 Complex in Maintenance DNA Methylation

Based on the current knowledge, we propose a simplified working model for the DNMT1/UHRF1 complex in maintenance DNA methylation (Figure 4): (i) The UHRF1 TTD interacts with PBR motif, and the SRA domain binds to the PHD domain. These intramolecular interactions lock UHRF1 in a closed conformation and keep it in an inactive state [48–52]. (ii) During replication, the UHRF1 SRA domain preferentially binds to hemi-methylated DNA, which triggers a conformational change in UHRF1 to dissociate the PHD finger from SRA and release TTD from the PBR motif, allowing TTD-PHD to interact with unmodified H3R2 and H3K9me2/3 marks. This process could be facilitated by the binding of USP7 or PIP5 to PBR. Multivalent engagement of chromatin modifications by various modules of UHRF1 would shift its conformation into an open state in which UHRF1’s E3 ubiquitin ligase activity is greatly stimulated, hence, UHRF1 can effectively add mono-ubiquitin to multiple lysine residues of histone H3. (iii) Through the physical interaction with a number of partner proteins such as PCNA, UHRF1 and ubiquitinated histone H3, DNMT1 is loaded onto chromatin and ready to catalyze methylation on hemi-methylated DNA. The UHRF1-dependent ubiquitination of histone H3 dramatically enhances its interaction with the DNMT1 RFTS domain and displaces the TTD-PHD module of UHRF1 from histone H3. Binding of dual mono-ubiquitinated H3 to DNMT1 will disrupt the intramolecular interaction of RFTS with the C-terminal catalytic region and allow the hemi-methylated DNA substrate to access the catalytic center. (iv) After a hemi-methylated CpG is converted into the fully methylated state, DNMT1 dissociates from the ubiquitinated histone H3 and moves along newly replicated DNA to carry out processive methylation reactions. Again, UHRF1 binds to hemi-methylated DNA first and adds ubiquitin marks to neighboring histone H3. At the same time, the deubiquitinase USP7 is activated to erase ubiquitin moieties from histone H3 at methyl DNA recovered regions [64]. Likely de-ubiquitination promotes the release of DNMT1 from histone H3 to ensure processive methylation reaction to proceed.

Genes 2018, 9, 600 7 of 12

Figure 4. A simplified model for the function of DNMT1/UHRF1 complex in maintenance DNA methylation. (i) UHRF1 adopts a closed conformation in which TTD binds to the PBR motif between SRA and RING finger. (ii) A conformational switch in UHRF1 from inactive closed conformation to active open state is triggered by hemi-methylated DNA binding (and may be facilitated by USP7 or PI5P binding to PBR), which allows TTD-PHD bind to H3K9me2/3 and the RING finger to add ubiquitin to multiple lysine residues of histone H3. (iii) Binding of RFTS to two mono-ubiquitinated histone H3 disrupts the interaction between RFTS and the C-terminal catalytic domain, causing a conformational change in DNMT1 that allows hemi-methylated DNA to access the catalytic center. (iv) After the DNA substrate is fully methylated, DNMT1 is released from the ubiquitinated histone H3 to allow processive DNA methylation along the newly replicated DNA. Again, UHRF1 binds to hemi-methylated DNA first and adds ubiquitin marks to the neighboring histone H3. At the same time, the deubiquitinase USP7 is activated through a currently unknown mechanism to erase ubiquitin marks from histone H3 at methyl DNA fully recovered regions. Likely de-ubiquitination promotes the release of DNMT1 from histone H3 to ensure processive methylation reaction to proceed.

9. Future Perspective

The past several years have witnessed tremendous advances in our understanding of the regulatory mechanisms involved in maintenance DNA methylation. However, many questions remain to be addressed due to the complex interaction network of DNMT1, UHRF1 and other partner factors. UHRF1-dependent histone H3 ubiquitination is critical for maintenance DNA methylation, but it seems that ubiquitinated histone H3 exists transiently during replication, and USP7 has been suggested to be a histone H3 deubiquitinase [64]. It was previously demonstrated that USP7 promotes the stability of DNMT1 and UHRF1 through preventing two proteins from polyubiquitination and subsequent proteasomal degradation [65–67], while the role of USP7 in this process is still under debate. A recent study provides evidence suggesting USP7 is not required for DNMT1 stability and the interaction between USP7 and DNMT1 is unlikely to play a major role in DNMT1 homeostasis [68]. Nonetheless, a number of studies showed that USP7 physically interacts with DNMT1 and UHRF1 [51,66,69]. Binding of USP7 stimulates DNMT1 enzymatic activity in vitro [69]. Moreover, USP7 deubiquitinates the ubiquitinated histone H3 in vitro. Inhibition or depletion of USP7 causes accumulation of ubiquitinated histone H3 and compromises maintenance DNA methylation [64]. Given that the enzymatic activity of USP7

Genes 2018, 9, 600 8 of 12

is allosterically regulated through the interaction with other partner proteins [70], it is important to determine how USP7 enzymatic activity is dynamically regulated during the process.

Recent evidence indicates that DNMT1 RFTS domain is a versatile protein interaction module. It has been demonstrated that DNMT1 RFTS domain interacts with the SRA domain and the interaction could stimulate DNMT1 enzymatic activity [29,35,69,71]. More recent studies found that the RFTS domain can interact with histone H3 N-terminal tail [27], but it binds much stronger to two mono-ubiquitinated histone H3 [57]. Given that maintenance DNA methylation is a highly efficient and orchestrated process, so far, more than 20 proteins have been reported to interact with DNMT1 [72]. Therefore, it is likely that some of these proteins, maybe in ubiquitinated form, could interact with RFTS domain to stimulate DNMT1 enzymatic activity during replication.

Lastly, Karg et al. have recently reported PAF15 is a major substrate for mono-ubiquitination by UHRF1 [73]. Dynamic PAF15 ubiquitination has previously been implicated in the regulation of translesion DNA bypass [74]. Interestingly, PAF15 exists in a mono-ubiquitinated form during normal replication, it will be interesting to explore if PAF15 has a role in maintenance DNA methylation.

Funding: The research in CM Qian’s laboratory has been supported by Hong Kong Research Grants Council, General Research Fund 17160016, 17127715 and 17117514.

Acknowledgments: We thank Dr. Julian Tanner for critical reading of the manuscript.

Conflicts of Interest: The authors declare no conflict of interest.


1. Smith, Z.D.; Sindhu, C.; Meissner, A. Molecular features of cellular reprogramming and development. Nat. Rev. Mol. Cell Biol. 2016, 17, 139–154. [CrossRef]

2. Slotkin, R.K.; Martienssen, R. Transposable elements and the epigenetic regulation of the genome. Nat. Rev. Genet. 2007, 8, 272–285. [CrossRef] [PubMed]

3. Chang, S.C.; Tucker, T.; Thorogood, N.P.; Brown, C.J. Mechanisms of X-chromosome inactivation. Front. Biosci. 2006, 11, 852–866. [CrossRef] [PubMed]

4. Dodge, J.E.; Ramsahoye, B.H.; Wo, Z.G.; Okano, M.; Li, E. De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation. Gene 2002, 289, 41–48. [CrossRef]

5. Lister, R.; Pelizzola, M.; Dowen, R.H.; Hawkins, R.D.; Hon, G.; Tonti-Filippini, J.; Nery, J.R.; Lee, L.; Ye, Z.; Ngo, Q.M.; et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 2009, 462, 315–322. [CrossRef] [PubMed]

6. Deaton, A.M.; Bird, A. CpG islands and the regulation of transcription. Genes Dev. 2011, 25, 1010–1022. [CrossRef] [PubMed]

7. Okano, M.; Bell, D.W.; Haber, D.A.; Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999, 99, 247–257. [CrossRef]

8. Kaneda, M.; Okano, M.; Hata, K.; Sado, T.; Tsujimoto, N.; Li, E.; Sasaki, H. Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 2004, 429, 900–903. [CrossRef]

9. Laurent, L.; Wong, E.; Li, G.; Huynh, T.; Tsirigos, A.; Ong, C.T.; Low, H.M.; Kin Sung, K.W.; Rigoutsos, I.; Loring, J.; et al. Dynamic changes in the human methylome during differentiation. Genome Res. 2010, 20, 320–331. [CrossRef]

10. Elliott, E.N.; Sheaffer, K.L.; Kaestner, K.H. The ‘de novo’ DNA methyltransferase Dnmt3b compensates the Dnmt1-deficient intestinal epithelium. eLife 2016, 5, e12975. [CrossRef]

11. Zemach, A.; McDaniel, I.E.; Silva, P.; Zilberman, D. Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 2010, 328, 916–919. [CrossRef] [PubMed]

12. Liang, G.; Chan, M.F.; Tomigahara, Y.; Tsai, Y.C.; Gonzales, F.A.; Li, E.; Laird, P.W.; Jones, P.A. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol. Cell. Biol. 2002, 22, 480–491. [CrossRef] [PubMed]

13. Ono, R.; Taki, T.; Taketani, T.; Taniwaki, M.; Kobayashi, H.; Hayashi, Y. LCX, leukemia-associated protein with a CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dysplasia having t(10;11)(q22;q23). Cancer Res. 2002, 62, 4075–4080. [PubMed]

Genes 2018, 9, 600 9 of 12

14. Lorsbach, R.B.; Moore, J.; Mathew, S.; Raimondi, S.C.; Mukatira, S.T.; Downing, J.R. TET1, a member of a novel protein family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;q23). Leukemia 2003, 17, 637–641. [CrossRef] [PubMed]

15. Bostick, M.; Kim, J.K.; Esteve, P.O.; Clark, A.; Pradhan, S.; Jacobsen, S.E. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 2007, 317, 1760–1764. [CrossRef] [PubMed]

16. Sharif, J.; Muto, M.; Takebayashi, S.; Suetake, I.; Iwamatsu, A.; Endo, T.A.; Shinga, J.; Mizutani-Koseki, Y.; Toyoda, T.; Okamura, K.; et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 2007, 450, 908–912. [CrossRef] [PubMed]

17. Von Meyenn, F.; Iurlaro, M.; Habibi, E.; Liu, N.Q.; Salehzadeh-Yazdi, A.; Santos, F.; Petrini, E.; Milagre, I.; Yu, M.; Xie, Z.; et al. Impairment of DNA methylation maintenance is the main cause of global demethylation in naive embryonic stem cells. Mol. Cell 2016, 62, 848–861. [CrossRef]

18. Chuang, L.S.; Ian, H.I.; Koh, T.W.; Ng, H.H.; Xu, G.; Li, B.F. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 1997, 277, 1996–2000. [CrossRef]

19. Rountree, M.R.; Bachman, K.E.; Baylin, S.B. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat. Genet. 2000, 25, 269–277. [CrossRef]

20. Ding, F.; Chaillet, J.R. In vivo stabilization of the Dnmt1 (cytosine-5)- methyltransferase protein. Proc. Natl. Acad. Sci. USA 2002, 99, 14861–14866. [CrossRef]

21. Pradhan, M.; Esteve, P.O.; Chin, H.G.; Samaranayke, M.; Kim, G.D.; Pradhan, S. CXXC domain of human DNMT1 is essential for enzymatic activity. Biochemistry 2008, 47, 10000–10009. [CrossRef] [PubMed]

22. Song, J.; Rechkoblit, O.; Bestor, T.H.; Patel, D.J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 2011, 331, 1036–1040. [CrossRef]

23. Zhang, Z.M.; Liu, S.; Lin, K.; Luo, Y.; Perry, J.J.; Wang, Y.; Song, J. Crystal structure of human DNA methyltransferase 1. J. Mol. Biol. 2015, 427, 2520–2531. [CrossRef] [PubMed]

24. Arita, K.; Isogai, S.; Oda, T.; Unoki, M.; Sugita, K.; Sekiyama, N.; Kuwata, K.; Hamamoto, R.; Tochio, H.; Sato, M.; et al. Recognition of modification status on a histone H3 tail by linked histone reader modules of the epigenetic regulator UHRF1. Proc. Natl. Acad. Sci. USA 2012, 109, 12950–12955. [CrossRef]

25. Avvakumov, G.V.; Walker, J.R.; Xue, S.; Li, Y.; Duan, S.; Bronner, C.; Arrowsmith, C.H.; Dhe-Paganon, S. Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1. Nature 2008, 455, 822–825. [CrossRef] [PubMed]

26. Takeshita, K.; Suetake, I.; Yamashita, E.; Suga, M.; Narita, H.; Nakagawa, A.; Tajima, S. Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1). Proc. Natl. Acad. Sci. USA 2011, 108, 9055–9059. [CrossRef]

27. Misaki, T.; Yamaguchi, L.; Sun, J.; Orii, M.; Nishiyama, A.; Nakanishi, M. The replication foci targeting sequence (RFTS) of DNMT1 functions as a potent histone H3 binding domain regulated by autoinhibition. Biochem. Biophys. Res. Commun. 2016, 470, 741–747. [CrossRef]

28. Bashtrykov, P.; Rajavelu, A.; Hackner, B.; Ragozin, S.; Carell, T.; Jeltsch, A. Targeted mutagenesis results in an activation of DNA methyltransferase 1 and confirms an autoinhibitory role of its RFTS domain. ChemBioChem 2014, 15, 743–748. [CrossRef] [PubMed]

29. Berkyurek, A.C.; Suetake, I.; Arita, K.; Takeshita, K.; Nakagawa, A.; Shirakawa, M.; Tajima, S. The DNA methyltransferase Dnmt1 directly interacts with the SET and RING finger-associated (SRA) domain of the multifunctional protein Uhrf1 to facilitate accession of the catalytic center to hemi-methylated DNA. J. Biol. Chem. 2014, 289, 379–386. [CrossRef]

30. Garvilles, R.G.; Hasegawa, T.; Kimura, H.; Sharif, J.; Muto, M.; Koseki, H.; Takahashi, S.; Suetake, I.; Tajima, S. Dual functions of the RFTS domain of Dnmt1 in replication-coupled DNA methylation and in protection of the genome from aberrant methylation. PLoS ONE 2015, 10, e0137509. [CrossRef] [PubMed]

31. Song, J.K.; Teplova, M.; Ishibe-Murakami, S.; Patel, D.J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 2012, 335, 709–712. [CrossRef] [PubMed]

32. Hashimoto, H.; Horton, J.R.; Zhang, X.; Bostick, M.; Jacobsen, S.E.; Cheng, X. The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix. Nature 2008, 455, 826–829. [CrossRef] [PubMed]

33. Arita, K.; Ariyoshi, M.; Tochio, H.; Nakamura, Y.; Shirakawa, M. Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism. Nature 2008, 455, 818–821. [CrossRef] [PubMed]

34. Qian, C.; Li, S.; Jakoncic, J.; Zeng, L.; Walsh, M.J.; Zhou, M.M. Structure and hemimethylated CpG binding of the SRA domain from human UHRF1. J. Biol. Chem. 2008, 283, 34490–34494. [CrossRef] [PubMed]

Genes 2018, 9, 600 10 of 12

35. Bashtrykov, P.; Jankevicius, G.; Jurkowska, R.Z.; Ragozin, S.; Jeltsch, A. The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism. J. Biol. Chem. 2014, 289, 4106–4115. [CrossRef] [PubMed]

36. Nady, N.; Lemak, A.; Walker, J.R.; Avvakumov, G.V.; Kareta, M.S.; Achour, M.; Xue, S.; Duan, S.; Allali-Hassani, A.; Zuo, X.; et al. Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein. J. Biol. Chem. 2011, 286, 24300–24311. [CrossRef]

37. Rajakumara, E.; Wang, Z.; Ma, H.; Hu, L.; Chen, H.; Lin, Y.; Guo, R.; Wu, F.; Li, H.; Lan, F.; et al. PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression. Mol. Cell 2011, 43, 275–284. [CrossRef]

38. Hu, L.; Li, Z.; Wang, P.; Lin, Y.; Xu, Y. Crystal structure of PHD domain of UHRF1 and insights into recognition of unmodified histone H3 arginine residue 2. Cell Res. 2011, 21, 1374–1378. [CrossRef]

39. Wang, C.; Shen, J.; Yang, Z.; Chen, P.; Zhao, B.; Hu, W.; Lan, W.; Tong, X.; Wu, H.; Li, G.; et al. Structural basis for site-specific reading of unmodified R2 of histone H3 tail by UHRF1 PHD finger. Cell Res. 2011, 21, 1379–1382. [CrossRef]

40. Xie, S.; Jakoncic, J.; Qian, C. UHRF1 double tudor domain and the adjacent PHD finger act together to recognize K9me3-containing histone H3 tail. J. Mol. Biol. 2012, 415, 318–328. [CrossRef]

41. Rothbart, S.B.; Krajewski, K.; Nady, N.; Tempel, W.; Xue, S.; Badeaux, A.I.; Barsyte-Lovejoy, D.; Martinez, J.Y.; Bedford, M.T.; Fuchs, S.M.; et al. Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation. Nat. Struct. Mol. Biol. 2012, 19, 1155–1160. [CrossRef] [PubMed]

42. Liu, X.; Gao, Q.; Li, P.; Zhao, Q.; Zhang, J.; Li, J.; Koseki, H.; Wong, J. UHRF1 targets DNMT1 for DNA methylation through cooperative binding of hemi-methylated DNA and methylated H3K9. Nat. Commun. 2013, 4, 1563. [CrossRef] [PubMed]

43. Rothbart, S.B.; Dickson, B.M.; Ong, M.S.; Krajewski, K.; Houliston, S.; Kireev, D.B.; Arrowsmith, C.H.; Strahl, B.D. Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation. Genes Dev. 2013, 27, 1288–1298. [CrossRef] [PubMed]

44. Citterio, E.; Papait, R.; Nicassio, F.; Vecchi, M.; Gomiero, P.; Mantovani, R.; Di Fiore, P.P.; Bonapace, I.M. Np95 is a histone-binding protein endowed with ubiquitin ligase activity. Mol. Cell. Biol. 2004, 24, 2526–2535. [CrossRef] [PubMed]

45. Karagianni, P.; Amazit, L.; Qin, J.; Wong, J. ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation. Mol. Cell. Biol. 2008, 28, 705–717. [CrossRef] [PubMed]

46. Nishiyama, A.; Yamaguchi, L.; Sharif, J.; Johmura, Y.; Kawamura, T.; Nakanishi, K.; Shimamura, S.; Arita, K.; Kodama, T.; Ishikawa, F.; et al. Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication. Nature 2013, 502, 249–253. [CrossRef] [PubMed]

47. Qin, W.; Wolf, P.; Liu, N.; Link, S.; Smets, M.; La Mastra, F.; Forne, I.; Pichler, G.; Horl, D.; Fellinger, K.; et al. DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination. Cell Res. 2015, 25, 911–929. [CrossRef] [PubMed]

48. Gelato, K.A.; Tauber, M.; Ong, M.S.; Winter, S.; Hiragami-Hamada, K.; Sindlinger, J.; Lemak, A.; Bultsma, Y.; Houliston, S.; Schwarzer, D.; et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol. Cell 2014, 54, 905–919. [CrossRef]

49. Fang, J.; Cheng, J.; Wang, J.; Zhang, Q.; Liu, M.; Gong, R.; Wang, P.; Zhang, X.; Feng, Y.; Lan, W.; et al. Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its histone recognition. Nat. Commun. 2016, 7, 11197. [CrossRef]

50. Harrison, J.S.; Cornett, E.M.; Goldfarb, D.; DaRosa, P.A.; Li, Z.M.; Yan, F.; Dickson, B.M.; Guo, A.H.; Cantu, D.V.; Kaustov, L.; et al. Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1. eLife 2016, 5, e17101. [CrossRef]

51. Zhang, Z.M.; Rothbart, S.B.; Allison, D.F.; Cai, Q.; Harrison, J.S.; Li, L.; Wang, Y.; Strahl, B.D.; Wang, G.G.; Song, J. An allosteric interaction links USP7 to deubiquitination and chromatin targeting of UHRF1. Cell Rep. 2015, 12, 1400–1406. [CrossRef] [PubMed]

52. Gao, L.; Tan, X.F.; Zhang, S.; Wu, T.; Zhang, Z.M.; Ai, H.W.; Song, J. An intramolecular interaction of UHRF1 reveals dual control for Its histone association. Structure 2018, 26, 304–311. [CrossRef] [PubMed]

53. Ferry, L.; Fournier, A.; Tsusaka, T.; Adelmant, G.; Shimazu, T.; Matano, S.; Kirsh, O.; Amouroux, R.; Dohmae, N.; Suzuki, T.; et al. Methylation of DNA ligase 1 by G9a/GLP recruits UHRF1 to replicating DNA and regulates DNA methylation. Mol. Cell 2017, 67, 550–565. [CrossRef] [PubMed]

Genes 2018, 9, 600 11 of 12

54. Vaughan, R.M.; Dickson, B.M.; Whelihan, M.F.; Johnstone, A.L.; Cornett, E.M.; Cheek, M.A.; Ausherman, C.A.; Cowles, M.W.; Sun, Z.W.; Rothbart, S.B. Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1. Proc. Natl. Acad. Sci. USA 2018, 115, 8775–8780. [CrossRef] [PubMed]

55. DaRosa, P.A.; Harrison, J.S.; Zelter, A.; Davis, T.N.; Brzovic, P.; Kuhlman, B.; Klevit, R.E. A Bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation. Mol. Cell 2018, 72, 753–765. [CrossRef] [PubMed]

56. Foster, B.M.; Stolz, P.; Mulholland, C.B.; Montoya, A.; Kramer, H.; Bultmann, S.; Bartke, T. Critical role of the UBL domain in stimulating the E3 ubiquitin ligase activity of UHRF1 toward chromatin. Mol. Cell 2018, 72, 739–752. [CrossRef] [PubMed]

57. Ishiyama, S.; Nishiyama, A.; Saeki, Y.; Moritsugu, K.; Morimoto, D.; Yamaguchi, L.; Arai, N.; Matsumura, R.; Kawakami, T.; Mishima, Y.; 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, 350–360. [CrossRef]

58. Li, T.; Wang, L.; Du, Y.; Xie, S.; Yang, X.; Lian, F.; Zhou, Z.; Qian, C. Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation. Nucleic Acids Res. 2018, 46, 3218–3231. [CrossRef]

59. Zhao, Q.; Zhang, J.; Chen, R.; Wang, L.; Li, B.; Cheng, H.; Duan, X.; Zhu, H.; Wei, W.; Li, J.; et al. Dissecting the precise role of H3K9 methylation in crosstalk with DNA maintenance methylation in mammals. Nat. Commun. 2016, 7, 12464. [CrossRef]

60. Cheng, J.; Yang, Y.; Fang, J.; Xiao, J.; Zhu, T.; Chen, F.; Wang, P.; Li, Z.; Yang, H.; Xu, Y. Structural insight into coordinated recognition of trimethylated histone H3 lysine 9 (H3K9me3) by the plant homeodomain (PHD) and tandem tudor domain (TTD) of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) protein. J. Biol. Chem. 2013, 288, 1329–1339. [CrossRef]

61. Veland, N.; Hardikar, S.; Zhong, Y.; Gayatri, S.; Dan, J.; Strahl, B.D.; Rothbart, S.B.; Bedford, M.T.; Chen, T. The arginine methyltransferase PRMT6 regulates DNA methylation and contributes to global DNA hypomethylation in cancer. Cell Rep. 2017, 21, 3390–3397. [CrossRef] [PubMed]

62. Sirbu, B.M.; Couch, F.B.; Feigerle, J.T.; Bhaskara, S.; Hiebert, S.W.; Cortez, D. Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev. 2011, 25, 1320–1327. [CrossRef] [PubMed]

63. Robertson, K.D.; Ait-Si-Ali, S.; Yokochi, T.; Wade, P.A.; Jones, P.L.; Wolffe, A.P. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat. Genet. 2000, 25, 338–342. [CrossRef] [PubMed]

64. Yamaguchi, L.; Nishiyama, A.; Misaki, T.; Johmura, Y.; Ueda, J.; Arita, K.; Nagao, K.; Obuse, C.; Nakanishi, M. Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation. Sci. Rep. 2017, 7, 55. [CrossRef] [PubMed]

65. Du, Z.; Song, J.; Wang, Y.; Zhao, Y.; Guda, K.; Yang, S.; Kao, H.Y.; Xu, Y.; Willis, J.; Markowitz, S.D.; et al. DNMT1 stability is regulated by proteins coordinating deubiquitination and acetylation-driven ubiquitination. Sci. Signal. 2010, 3, ra80. [CrossRef] [PubMed]

66. Cheng, J.; Yang, H.; Fang, J.; Ma, L.; Gong, R.; Wang, P.; Li, Z.; Xu, Y. Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation. Nat. Commun. 2015, 6, 7023. [CrossRef] [PubMed]

67. Ma, H.; Chen, H.; Guo, X.; Wang, Z.; Sowa, M.E.; Zheng, L.; Hu, S.; Zeng, P.; Guo, R.; Diao, J.; et al. M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability. Proc. Natl. Acad. Sci. USA 2012, 109, 4828–4833. [CrossRef]

68. Yarychkivska, O.; Tavana, O.; Gu, W.; Bestor, T.H. Independent functions of DNMT1 and USP7 at replication foci. Epigenet. Chromatin 2018, 11, 9. [CrossRef]

69. Felle, M.; Joppien, S.; Nemeth, A.; Diermeier, S.; Thalhammer, V.; Dobner, T.; Kremmer, E.; Kappler, R.; Langst, G. The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1. Nucleic Acids Res. 2011, 39, 8355–8365. [CrossRef]

70. Kim, R.Q.; Sixma, T.K. Regulation of USP7: A high incidence of E3 complexes. J. Mol. Biol. 2017, 429, 3395–3408. [CrossRef]

71. Achour, M.; Jacq, X.; Ronde, P.; Alhosin, M.; Charlot, C.; Chataigneau, T.; Jeanblanc, M.; Macaluso, M.; Giordano, A.; Hughes, A.D.; et al. The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression. Oncogene 2008, 27, 2187–2197. [CrossRef] [PubMed]

Genes 2018, 9, 600 12 of 12

72. Qin, W.; Leonhardt, H.; Pichler, G. Regulation of DNA methyltransferase 1 by interactions and modifications. Nucleus 2011, 2, 392–402. [CrossRef]

73. Karg, E.; Smets, M.; Ryan, J.; Forne, I.; Qin, W.; Mulholland, C.B.; Kalideris, G.; Imhof, A.; Bultmann, S.; Leonhardt, H. Ubiquitome analysis reveals PCNA-associated factor 15 (PAF15) as a specific ubiquitination target of UHRF1 in embryonic stem cells. J. Mol. Biol. 2017, 429, 3814–3824. [CrossRef] [PubMed]

74. Povlsen, L.K.; Beli, P.; Wagner, S.A.; Poulsen, S.L.; Sylvestersen, K.B.; Poulsen, J.W.; Nielsen, M.L.; Bekker-Jensen, S.; Mailand, N.; Choudhary, C. Systems-wide analysis of ubiquitylation dynamics reveals a key role for PAF15 ubiquitylation in DNA-damage bypass. Nat. Cell Biol. 2012, 14, 1089–1098. [CrossRef] [PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

  • Introduction
  • DNMT1 is an Autoinhibited DNA Methyltransferase
  • UHRF1 is Required for Proper Loading of DNMT1 onto Chromatin
  • Allosteric Regulation of UHRF1 E3 Ubiquitin Ligase Activity
  • UHRF1-Dependent Histone H3 Ubiquitination Stimulates DNMT1 Enzymatic Activity
  • DNMT1 Recruitment and Stimulation by UHRF1 N-Terminal UBL Domain
  • Epigenetic Crosstalk between DNA Methylation and Histone Modifications in Maintenance DNA Methylation
  • A Working Model for DNMT1/UHRF1 Complex in Maintenance DNA Methylation
  • Future Perspective
  • References