The colorful history of active DNA demethylation.

Patterns of DNA cytosine methylation are subject to mitotic inheritance in both plants and vertebrates. Plants use 5-methylcytosine glycosylases and the base excision repair pathway to remove excess cytosine methylation. In mammals, active demethylation has been proposed to operate via several very different mechanisms. Two recent reports in Nature now claim that the demethylation process is initiated by the same enzymes that establish the methylation mark, the DNA methyltransferases DNMT3A and DNMT3B (Kangaspeska et al., 2008; Métivier et al., 2008).

Methylation-directed glycosylation of chromatin factors represses retrotransposon promoters.

The mechanisms by which methylated mammalian promoters are transcriptionally silenced even in the presence of all of the factors required for their expression have long been a major unresolved issue in the field of epigenetics. Repression requires the assembly of a methylation-dependent silencing complex that contains the TRIM28 protein (also known as KAP1 and TIF1ß), a scaffolding protein without intrinsic repressive or DNA-binding properties. The identity of the key effector within this complex that represses transcription is unknown. We developed a methylation-sensitized interaction screen which revealed that TRIM28 was complexed with O-linked ß-N-acetylglucosamine transferase (OGT) only in cells that had normal genomic methylation patterns. OGT is the only glycosyltransferase that modifies cytoplasmic and nuclear protein by transfer of N-acetylglucosamine (O-GlcNAc) to serine and threonine hydroxyls. Whole-genome analysis showed that O-glycosylated proteins and TRIM28 were specifically bound to promoters of active retrotransposons and to imprinting control regions, the two major regulatory sequences controlled by DNA methylation. Furthermore, genome-wide loss of DNA methylation caused a loss of O-GlcNAc from multiple transcriptional repressor proteins associated with TRIM28. A newly developed Cas9-based editing method for targeted removal of O-GlcNAc was directed against retrotransposon promoters. Local chromatin de-GlcNAcylation specifically reactivated the expression of the targeted retrotransposon family without loss of DNA methylation. These data revealed that O-linked glycosylation of chromatin factors is essential for the transcriptional repression of methylated retrotransposons.

BAH domains and a histone-like motif in DNA methyltransferase 1 (DNMT1) regulate de novo and maintenance methylation in vivo.

DNA methyltransferase 1 (DNMT1) is a multidomain protein believed to be involved only in the passive transmission of genomic methylation patterns via maintenance methylation. The mechanisms that regulate DNMT1 activity and targeting are complex and poorly understood. We used embryonic stem (ES) cells to investigate the function of the uncharacterized bromo-adjacent homology (BAH) domains and the glycine-lysine (GK) repeats that join the regulatory and catalytic domains of DNMT1. We removed the BAH domains by means of a CRISPR/Cas9-mediated deletion within the endogenous Dnmt1 locus. The internally deleted protein failed to associate with replication foci during S phase in vivo and lost the ability to mediate maintenance methylation. The data indicate that ablation of the BAH domains causes DNMT1 to be excluded from replication foci even in the presence of the replication focus-targeting sequence (RFTS). The GK repeats resemble the N-terminal tails of histones H2A and H4 and are normally acetylated. Substitution of lysines within the GK repeats with arginines to prevent acetylation did not alter the maintenance activity of DNMT1 but unexpectedly activated de novo methylation of paternal imprinting control regions (ICRs) in mouse ES cells; maternal ICRs remained unmethylated. We propose a model under which DNMT1 deposits paternal imprints in male germ cells in an acetylation-dependent manner. These data reveal that DNMT1 responds to multiple regulatory inputs that control its localization as well as its activity and is not purely a maintenance methyltransferase but can participate in the de novo methylation of a small but essential compartment of the genome.

Photochemical conversion of a cytidine derivative to a thymidine analog via [2+2]-cycloaddition.

Epigenetic information is encoded in the mammalian genome in the form of cytosines methylated at the 5 position. Cytosine methylation has multiple biological effects, but our understanding of these effects has lagged because extant methods for mapping methylation sites genome-wide have severe shortcomings. For instance, the gold standard bisulfite sequencing approach suffers from the use of harsh reaction conditions resulting in DNA cleavage and incomplete conversion of unmethylated cytosine to uracil. We report here on a new photochemical method in which a DNA (cytosine-5)-methyltransferase can be used to covalently attach reactive functionalities which upon irradiation at ∼350 nm initiate photoinduced intramolecular reactions that convert modified C to T analogues. We synthesized a model compound, a cinnamyl ether-containing cytidine derivative, and demonstrated its conversion to a thymidine analogue using mild conditions and a DNA-compatible wavelength (∼350 nm), enabled by the use of a triplet sensitizer, thioxanthone. Transfer of a cinnamyl ether or comparable reactive functionality from an AdoMet analog to cytosine followed by the use of this photoconversion method would require only small amounts of DNA and allow complete methylation profiling on both long and short read sequencing platforms.

Notes on the role of dynamic DNA methylation in mammalian development.

It has been nearly 40 y since it was suggested that genomic methylation patterns could be transmitted via maintenance methylation during S phase and might play a role in the dynamic regulation of gene expression during development [Holliday R, Pugh JE (1975) Science 187(4173):226-232; Riggs AD (1975) Cytogenet Cell Genet 14(1):9-25]. This revolutionary proposal was justified by "... our almost complete ignorance of the mechanism for the unfolding of the genetic program during development" that prevailed at the time. Many correlations between transcriptional activation and demethylation have since been reported, but causation has not been demonstrated and to date there is no reasonable proof of the existence of a complex biochemical system that activates and represses genes via reversible DNA methylation. Such a system would supplement or replace the conserved web of transcription factors that regulate cellular differentiation in organisms that have unmethylated genomes (such as Caenorhaditis elegans and the Dipteran insects) and those that methylate their genomes. DNA methylation does have essential roles in irreversible promoter silencing, as in the monoallelic expression of imprinted genes, in the silencing of transposons, and in X chromosome inactivation in female mammals. Rather than reinforcing or replacing regulatory pathways that are conserved between organisms that have either methylated or unmethylated genomes, DNA methylation endows genomes with the ability to subject specific sequences to irreversible transcriptional silencing even in the presence of all of the factors required for their expression, an ability that is generally unavailable to organisms that have unmethylated genomes.

Protein O-Glucosyltransferase 1 (POGLUT1) Promotes Mouse Gastrulation through Modification of the Apical Polarity Protein CRUMBS2.

Crumbs family proteins are apical transmembrane proteins with ancient roles in cell polarity. Mouse Crumbs2 mutants arrest at midgestation with abnormal neural plate morphology and a deficit of mesoderm caused by defects in gastrulation. We identified an ENU-induced mutation, wsnp, that phenocopies the Crumbs2 null phenotype. We show that wsnp is a null allele of Protein O-glucosyltransferase 1 (Poglut1), which encodes an enzyme previously shown to add O-glucose to EGF repeats in the extracellular domain of Drosophila and mammalian Notch, but the role of POGLUT1 in mammalian gastrulation has not been investigated. As predicted, we find that POGLUT1 is essential for Notch signaling in the early mouse embryo. However, the loss of mouse POGLUT1 causes an earlier and more dramatic phenotype than does the loss of activity of the Notch pathway, indicating that POGLUT1 has additional biologically relevant substrates. Using mass spectrometry, we show that POGLUT1 modifies EGF repeats in the extracellular domain of full-length mouse CRUMBS2. CRUMBS2 that lacks the O-glucose modification fails to be enriched on the apical plasma membrane and instead accumulates in the endoplasmic reticulum. The data demonstrate that CRUMBS2 is the target of POGLUT1 for the gastrulation epithelial-to-mesenchymal transitions (EMT) and that all activity of CRUMBS2 depends on modification by POGLUT1. Mutations in human POGLUT1 cause Dowling-Degos Disease, POGLUT1 is overexpressed in a variety of tumor cells, and mutations in the EGF repeats of human CRUMBS proteins are associated with human congenital nephrosis, retinitis pigmentosa and retinal degeneration, suggesting that O-glucosylation of CRUMBS proteins has broad roles in human health.

Ectopic DNMT3L triggers assembly of a repressive complex for retroviral silencing in somatic cells.

UNLABELLED: Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransferase 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1ß and KAP1) in ES cells and orchestrates retroviral silencing activity with TRIM28 through mechanisms including, but not limited to, de novo DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney murine leukemia virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1γ), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late-passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly integrated retroviruses. IMPORTANCE: Almost half of the mammalian genome is composed of endogenous retroviruses and other retrotransposable elements that threaten genomic integrity. These elements are usually subject to epigenetic silencing. We discovered that two epigenetic regulators that lack enzymatic activity, DNA methyltransferase 3-like (DNMT3L) and tripartite motif-containing protein 28 (TRIM28), collaborate with each other to impose retroviral silencing. In addition to modulating de novo DNA methylation, we found that by interacting with TRIM28, DNMT3L can attract various enzymes to form a DNMT3L-induced repressive complex to remove active marks and add repressive marks to histone proteins. Collectively, these results reveal a novel and pivotal function of DNMT3L in shaping the chromatin modifications necessary for retroviral and retrotransposon silencing.

Chromatin and sequence features that define the fine and gross structure of genomic methylation patterns.

Abnormalities of genomic methylation patterns are lethal or cause disease, but the cues that normally designate CpG dinucleotides for methylation are poorly understood. We have developed a new method of methylation profiling that has single-CpG resolution and can address the methylation status of repeated sequences. We have used this method to determine the methylation status of >275 million CpG sites in human and mouse DNA from breast and brain tissues. Methylation density at most sequences was found to increase linearly with CpG density and to fall sharply at very high CpG densities, but transposons remained densely methylated even at higher CpG densities. The presence of histone H2A.Z and histone H3 di- or trimethylated at lysine 4 correlated strongly with unmethylated DNA and occurred primarily at promoter regions. We conclude that methylation is the default state of most CpG dinucleotides in the mammalian genome and that a combination of local dinucleotide frequencies, the interaction of repeated sequences, and the presence or absence of histone variants or modifications shields a population of CpG sites (most of which are in and around promoters) from DNA methyltransferases that lack intrinsic sequence specificity.

Decatenation checkpoint deficiency in stem and progenitor cells.

The decatenation checkpoint normally delays entry into mitosis until chromosomes have been disentangled through the action of topoisomerase II. We have found that the decatenation checkpoint is highly inefficient in mouse embryonic stem cells, mouse neural progenitor cells, and human CD34+ hematopoietic progenitor cells. Checkpoint efficiency increased when embryonic stem cells were induced to differentiate, which suggests that the deficiency is a feature of the undifferentiated state. Embryonic stem cells completed cell division in the presence of entangled chromosomes, which resulted in severe aneuploidy in the daughter cells. The decatenation checkpoint deficiency is likely to increase the rates of chromosome aberrations in progenitor cells, stem cells, and cancer stem cells.

DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA.

Mammals use DNA methylation for the heritable silencing of retrotransposons and imprinted genes and for the inactivation of the X chromosome in females. The establishment of patterns of DNA methylation during gametogenesis depends in part on DNMT3L, an enzymatically inactive regulatory factor that is related in sequence to the DNA methyltransferases DNMT3A and DNMT3B. The main proteins that interact in vivo with the product of an epitope-tagged allele of the endogenous Dnmt3L gene were identified by mass spectrometry as DNMT3A2, DNMT3B and the four core histones. Peptide interaction assays showed that DNMT3L specifically interacts with the extreme amino terminus of histone H3; this interaction was strongly inhibited by methylation at lysine 4 of histone H3 but was insensitive to modifications at other positions. Crystallographic studies of human DNMT3L showed that the protein has a carboxy-terminal methyltransferase-like domain and an N-terminal cysteine-rich domain. Cocrystallization of DNMT3L with the tail of histone H3 revealed that the tail bound to the cysteine-rich domain of DNMT3L, and substitution of key residues in the binding site eliminated the H3 tail-DNMT3L interaction. These data indicate that DNMT3L recognizes histone H3 tails that are unmethylated at lysine 4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.

The parental non-equivalence of imprinting control regions during mammalian development and evolution.

In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.

MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline.

Small RNAs associate with Argonaute proteins and serve as sequence-specific guides for regulation of mRNA stability, productive translation, chromatin organization, and genome structure. In animals, the Argonaute superfamily segregates into two clades. The Argonaute clade acts in RNAi and in microRNA-mediated gene regulation in partnership with 21-22 nt RNAs. The Piwi clade, and their 26-30 nt piRNA partners, have yet to be assigned definitive functions. In mice, two Piwi-family members have been demonstrated to have essential roles in spermatogenesis. Here, we examine the effects of disrupting the gene encoding the third family member, MIWI2. Miwi2-deficient mice display a meiotic-progression defect in early prophase of meiosis I and a marked and progressive loss of germ cells with age. These phenotypes may be linked to an inappropriate activation of transposable elements detected in Miwi2 mutants. Our observations suggest a conserved function for Piwi-clade proteins in the control of transposons in the germline.

Altered methylation patterns in cancer cell genomes: cause or consequence?

CpG islands are associated with at least half of all cellular genes and are normally methylation-free. Dense methylation of cytosine residues within islands causes strong and heritable transcriptional silencing. Such silencing normally occurs almost solely at genes subject to genomic imprinting or to X chromosome inactivation. Aberrant methylation of CpG islands associated with tumor suppressor genes has been proposed to contribute to carcinogenesis. However, questions of mechanisms underlying the cancer changes and the precise consequences for tumorigenesis exist in the field, and must continue to be addressed before the importance of abnormalities in genomic methylation patterns in carcinogenesis can be fully understood. In this article, two workers in DNA methylation, one concentrating on cancer biology and the other on developmental biology, address recurrent questions about cancer epigenetics from different perspectives. The goal is to highlight important controversies in the field which can be productive targets of ongoing and future research.

Cytosine methylation: remaining faithful.

DNA methyltransferase-1 (DNMT1) has a higher specific activity on hemimethylated DNA than on unmethylated DNA, but this preference is too small to explain the faithful mitotic inheritance of genomic methylation patterns. New genetic studies in plants and mammals have identified a novel factor that increases the fidelity of maintenance methylation.

Biological functions of DNA methyltransferase 1 require its methyltransferase activity.

DNA methyltransferase 1 (DNMT1) has been reported to interact with a wide variety of factors and to contain intrinsic transcriptional repressor activity. When a conservative point mutation was introduced at the key catalytic residue, mutant DNMT1 failed to rescue any of the phenotypes of Dnmt1-null embryonic stem (ES) cells, which indicated that the biological functions of DNMT1 are exerted through the methylation of DNA. ES cells that expressed the mutant protein did not survive differentiation. Intracisternal A-particle family retrotransposons were no longer methylated and were transcribed at high levels. The proper localization of DNMT1 depended on normal genomic methylation, and we discuss the implications of this finding for epigenetic dysregulation in cancer.

Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L.

Mammalian genomes employ heritable cytosine methylation in the long-term silencing of retrotransposons and genes subject to genomic imprinting and X chromosome inactivation. Little is known of the mechanisms that direct cytosine methylation to specific sequences. Here we show that DNA methyltransferase 3-like (Dnmt3L (ref. 1)) is expressed in testes during a brief perinatal period in the non-dividing precursors of spermatogonial stem cells at a stage where retrotransposons undergo de novo methylation. Deletion of the Dnmt3L gene prevented the de novo methylation of both long-terminal-repeat (LTR) and non-LTR retrotransposons, which were transcribed at high levels in spermatogonia and spermatocytes. Loss of Dnmt3L from early germ cells also caused meiotic failure in spermatocytes, which do not express Dnmt3L. Whereas dispersed repeated sequences were demethylated in mutant germ cells, tandem repeats in pericentric regions were methylated normally. This result indicates that the Dnmt3L protein might have a function in the de novo methylation of dispersed repeated sequences in a premeiotic genome scanning process that occurs in male germ cells at about the time of birth.

BRCA1 promoter methylation is associated with increased mortality among women with breast cancer.

Promoter-CpG island hypermethylation has been proposed as an alternative mechanism to inactivate BRCA1 in the breast where somatic mutations of BRCA1 are rare. To better understand breast cancer etiology and progression, we explored the association between BRCA1 promoter methylation status and prognostic factors as well as survival among women with breast cancer. Promoter methylation of BRCA1 was assessed in 851 archived tumor tissues collected from a population-based study of women diagnosed with invasive or in situ breast cancer in 1996-1997, and who were followed for vital status through the end of 2002. About 59% of the tumors were methylated at the promoter of BRCA1. The BRCA1 promoter methylation was more frequent in invasive cancers (P = 0.02) and among premenopausal cases (P = 0.05). BRCA1 promoter methylation was associated with increased risk of breast cancer-specific mortality (age-adjusted HR 1.71; 95% CI: 1.05-2.78) and all-cause mortality (age-adjusted HR 1.49; 95% CI: 1.02-2.18). Neither dietary methyl intakes in the year prior to the baseline interview nor the functional polymorphisms in one-carbon metabolism were associated with BRCA1 methylation status. Our study is the first epidemiological investigation on the prognostic value of BRCA1 promoter methylation in a large population-based cohort of breast cancer patients. Our results indicate that BRCA1 promoter methylation is an important factor to consider in predicting breast cancer survival.

Allele-specific demethylation at an imprinted mammalian promoter.

A screen for imprinted genes on mouse Chromosome 7 recently identified Inpp5f_v2, a paternally expressed retrogene lying within an intron of Inpp5f. Here, we identify a novel paternally expressed variant of the Inpp5f gene (Inpp5f_v3) that shows a number of unusual features. Inpp5f_v3 initiates from a CpG-rich repeat region adjoining two B1 elements, despite previous reports that SINEs are generally excluded from imprinted promoters. Accordingly, we find that the Inpp5f_v3 promoter acquires methylation around the time of implantation, when many repeat families undergo de novo epigenetic silencing. Methylation is then lost specifically on the paternally derived allele during the latter stages of embryonic development, resulting in imprinted transcriptional activation on the demethylated allele. Methylation analyses in embryos lacking maternal methylation imprints suggest that the primary imprinting mark resides within an intronic CpG island approximately 1 kb downstream of the Inpp5f_v3 transcriptional start site. These data support the hypothesis that SINEs can influence gene expression by attracting de novo methylation during development, a property likely to explain their exclusion from other imprinted promoters.