DNMT3L Modulates Significant and Distinct Flanking Sequence Preference for DNA Methylation by DNMT3A and DNMT3B In Vivo

The DNTM3A and DNMT3B de novo DNA methyltransferases (DNMTs) are responsible for setting genomic DNA methylation patterns, a key layer of epigenetic information. Here, using an in vivo episomal methylation assay and extensive bisulfite methylation sequencing, we show that human DNMT3A and DNMT3B possess significant and distinct flanking sequence preferences for target CpG sites. Selection for high or low efficiency sites is mediated by the base composition at the −2 and +2 positions flanking the CpG site for DNMT3A, and at the −1 and +1 positions for DNMT3B. This intrinsic preference reproducibly leads to the formation of specific de novo methylation patterns characterized by up to 34-fold variations in the efficiency of DNA methylation at individual sites. Furthermore, analysis of the distribution of signature methylation hotspot and coldspot motifs suggests that DNMT flanking sequence preference has contributed to shaping the composition of CpG islands in the human genome. Our results also show that the DNMT3L stimulatory factor modulates the formation of de novo methylation patterns in two ways. First, DNMT3L selectively focuses the DNA methylation machinery on properly chromatinized DNA templates. Second, DNMT3L attenuates the impact of the intrinsic DNMT flanking sequence preference by providing a much greater boost to the methylation of poorly methylated sites, thus promoting the formation of broader and more uniform methylation patterns. This study offers insights into the manner by which DNA methylation patterns are deposited and reveals a new level of interplay between members of the de novo DNMT family.     

AUF1 cell cycle variations define genomic DNA methylation by regulation of DNMT1 mRNA stability

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/degradation factor 1 (AUF1)/heterogeneous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3' untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell.     

DNMT1 is a component of a multiprotein DNA replication complex

DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase alpha activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.     

DNMT1 is a Component of a Multiprotein DNA Replication Complex

DNA methylation is a major determinant of epigenetic inheritance and plays an important role in genome stability. The accurate propagation of DNA methylation patterns with cell division requires that methylation be closely coupled to DNA replication, however the precise molecular determinants of this interaction have not been defined. In the present study, we show that the predominant DNA methyltransferase species in somatic cells, DNMT1, is a component of a multiprotein DNA replication complex termed the DNA synthesome that fully supports semi-conservative DNA replication in a cell-free system. DNMT1 protein and activity were found to co-purify with the human DNA synthesome through a series of subcellular fractionation and chromatography steps, resulting in an enrichment of methyltransferase specific activity from two human cell lines. DNA methyltransferase activity co-eluted with in vitro replication activity and DNA polymerase a activity on sucrose density gradients suggesting that DNMT1 is a tightly bound, core component of the replication complex. The synthesome-associated pool of DNA methyltransferase exhibited both maintenance and de novo methyltransferase activity and the ratio of the two was similar to that observed in whole cell lysates and for recombinant DNMT1. These data indicate that interactions within the synthesome complex do not influence the intrinsic preference of DNMT1 for hemimethylated DNA, but suggest that newly replicated DNA may be subject to low level de novo methylation. The data indicate that DNA methylation is tightly coupled to replication through physical interaction of DNMT1 and core components of the replication machinery. The definition of the molecular interactions between DNMT1 and other proteins in the replication complex in normal and neoplastic cells will provide further insight into the regulation of DNA methylation and the mechanisms underlying the alteration of DNA methylation patterns during carcinogenesis.     

Transient DNMT1 suppression reveals hidden heritable marks in the genome

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.     

Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation

DNA methylation is a central epigenetic modification in mammals, with essential roles in development and disease. De novo DNA methyltransferases establish DNA methylation patterns in specific regions within the genome by mechanisms that remain poorly understood. Here we show that protein citrullination by peptidylarginine deiminase 4 (PADI4) affects the function of the DNA methyltransferase DNMT3A. We found that DNMT3A and PADI4 interact, from overexpressed as well as untransfected cells, and associate with each other''s enzymatic activity. Both in vitro and in vivo, PADI4 was shown to citrullinate DNMT3A. We identified a sequence upstream of the PWWP domain of DNMT3A as its primary region citrullinated by PADI4. Increasing the PADI4 level caused the DNMT3A protein level to increase as well, provided that the PADI4 was catalytically active, and RNAi targeting PADI4 caused reduced DNMT3A levels. Accordingly, pulse-chase experiments revealed stabilization of the DNMT3A protein by catalytically active PADI4. Citrullination and increased expression of native DNMT3A by PADI4 were confirmed in PADI4-knockout MEFs. Finally, we showed that PADI4 overexpression increases DNA methyltransferase activity in a catalytic-dependent manner and use bisulfite pyrosequencing to demonstrate that PADI4 knockdown causes significant reduction of CpG methylation at the p21 promoter, a known target of DNMT3A and PADI4. Protein citrullination by PADI4 thus emerges as a novel mechanism for controlling a de novo DNA methyltransferase. Our results shed new light on how post-translational modifications might contribute to shaping the genomic CpG methylation landscape.     

Phosphorylation of human DNMT1: implication of cyclin-dependent kinases

DNA methylation plays a central role in the epigenetic regulation of gene expression during development and progression of cancer diseases. The inheritance of specific DNA methylation patterns are acquired in the early embryo and are specifically maintained after cellular replication via the DNA methyltransferase 1 (DNMT1). Recent studies have suggested that the enzymatic activity of DNMT1 is possibly modulated by phosphorylation of serine/threonine residues located in the N-terminal domain of the enzyme. In the present work, we report that cyclin-dependent kinases (CDKs) 1, 2 and 5 can phosphorylate Ser154 of human DNMT1 in vitro. Further evidence of phosphorylation of endogenous DNMT1 at position 154 by CDKs is also found in 293 cells treated with roscovitine, a specific inhibitor of CDK1, 2 and 5. To determine the importance of Ser154 phosphorylation, a mutant of DNMT1 encoding a single-point mutation at position 154 (S154A) was generated. This mutation induced a severe loss of enzymatic activity when compared to wild type DNMT1. Moreover, after treatment with 5-Aza-2′-Deoxycytidine (5-aza-dC), a faster decline in DNMT1 protein level was observed for HEK-293 cells expressing DNMT1(S154A) as compared to cells expressing wild type DNMT1. Our data suggest that phosphorylation of DNMT1 at Ser154 by CDKs is important for enzymatic activity and protein stability of DNMT1. Considering that tumour-associated cell cycle defects are often mediated by alterations in CDK activity, our results suggest that dysregulation of cell cycle via CDKs could induce abnormal phosphorylation of DNMT1 and lead to DNA hypermethylation often observed in cancer cells.     

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.     

A role for LSH in facilitating DNA methylation by DNMT1 through enhancing UHRF1 chromatin association

LSH, a SNF2 family DNA helicase, is a key regulator of DNA methylation in mammals. How LSH facilitates DNA methylation is not well defined. While previous studies with mouse embryonic stem cells (mESc) and fibroblasts (MEFs) derived from Lsh knockout mice have revealed a role of Lsh in de novo DNA methylation by Dnmt3a/3b, here we report that LSH contributes to DNA methylation in various cell lines primarily by promoting DNA methylation by DNMT1. We show that loss of LSH has a much bigger effect in DNA methylation than loss of DNMT3A and DNMT3B. Mechanistically, we demonstrate that LSH interacts with UHRF1 but not DNMT1 and facilitates UHRF1 chromatin association and UHRF1-catalyzed histone H3 ubiquitination in an ATPase activity-dependent manner, which in turn promotes DNMT1 recruitment to replication fork and DNA methylation. Notably, UHRF1 also enhances LSH association with the replication fork. Thus, our study identifies LSH as an essential factor for DNA methylation by DNMT1 and provides novel insight into how a feed-forward loop between LSH and UHRF1 facilitates DNMT1-mediated maintenance of DNA methylation in chromatin.     

Age and sun exposure-related widespread genomic blocks of hypomethylation in nonmalignant skin

BackgroundAging and sun exposure are the leading causes of skin cancer. It has been shown that epigenetic changes, such as DNA methylation, are well established mechanisms for cancer, and also have emerging roles in aging and common disease. Here, we directly ask whether DNA methylation is altered following skin aging and/or chronic sun exposure in humans.ResultsWe compare epidermis and dermis of both sun-protected and sun-exposed skin derived from younger subjects (under 35 years old) and older subjects (over 60 years old), using the Infinium HumanMethylation450 array and whole genome bisulfite sequencing. We observe large blocks of the genome that are hypomethylated in older, sun-exposed epidermal samples, with the degree of hypomethylation associated with clinical measures of photo-aging. We replicate these findings using whole genome bisulfite sequencing, comparing epidermis from an additional set of younger and older subjects. These blocks largely overlap known hypomethylated blocks in colon cancer and we observe that these same regions are similarly hypomethylated in squamous cell carcinoma samples.ConclusionsThese data implicate large scale epigenomic change in mediating the effects of environmental damage with photo-aging.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0644-y) contains supplementary material, which is available to authorized users.     

Aberrant signature methylome by DNMT1 hot spot mutation in hereditary sensory and autonomic neuropathy 1E

DNA methyltransferase 1 (DNMT1) is essential for DNA methylation, gene regulation and chromatin stability. We previously discovered DNMT1 mutations cause hereditary sensory and autonomic neuropathy type 1 with dementia and hearing loss (HSAN1E; OMIM 614116). HSAN1E is the first adult-onset neurodegenerative disorder caused by a defect in a methyltransferase gene. HSAN1E patients appear clinically normal until young adulthood, then begin developing the characteristic symptoms involving central and peripheral nervous systems. Some HSAN1E patients also develop narcolepsy and it has recently been suggested that HSAN1E is allelic to autosomal dominant cerebellar ataxia, deafness, with narcolepsy (ADCA-DN; OMIM 604121), which is also caused by mutations in DNMT1. A hotspot mutation Y495C within the targeting sequence domain of DNMT1 has been identified among HSAN1E patients. The mutant DNMT1 protein shows premature degradation and reduced DNA methyltransferase activity. Herein, we investigate genome-wide DNA methylation at single-base resolution through whole-genome bisulfite sequencing of germline DNA in 3 pairs of HSAN1E patients and their gender- and age-matched siblings. Over 1 billion 75-bp single-end reads were generated for each sample. In the 3 affected siblings, overall methylation loss was consistently found in all chromosomes with X and 18 being most affected. Paired sample analysis identified 564,218 differentially methylated CpG sites (DMCs; P < 0.05), of which 300?134 were intergenic and 264?084 genic CpGs. Hypomethylation was predominant in both genic and intergenic regions, including promoters, exons, most CpG islands, L1, L2, Alu, and satellite repeats and simple repeat sequences. In some CpG islands, hypermethylated CpGs outnumbered hypomethylated CpGs. In 201 imprinted genes, there were more DMCs than in non-imprinted genes and most were hypomethylated. Differentially methylated region (DMR) analysis identified 5649 hypomethylated and 1872 hypermethylated regions. Importantly, pathway analysis revealed 1693 genes associated with the identified DMRs were highly associated in diverse neurological disorders and NAD+/NADH metabolism pathways is implicated in the pathogenesis. Our results provide novel insights into the epigenetic mechanism of neurodegeneration arising from a hotspot DNMT1 mutation and reveal pathways potentially important in a broad category of neurological and psychological disorders.     

CB1‐receptor knockout neonatal mice are protected against ethanol‐induced impairments of DNMT1, DNMT3A,and DNA methylation

The significant consequences of ethanol use during pregnancy are neurobehavioral abnormalities involving hippocampal and neocortex malfunctions that cause learning and memory deficits collectively named fetal alcohol spectrum disorder. However, the molecular mechanisms underlying these abnormalities are still poorly understood and therefore warrant systematic research. Here, we document novel epigenetic abnormalities in the mouse model of fetal alcohol spectrum disorder. Ethanol treatment of P7 mice, which induces activation of caspase 3, impaired DNA methylation through reduced DNA methyltransferases (DNMT1 and DNMT3A) levels. Inhibition of caspase 3 activity, before ethanol treatment, rescued DNMT1, DNMT3A proteins as well as DNA methylation levels. Blockade of histone methyltransferase (G9a) activity or cannabinoid receptor type‐1 (CB1R), prior to ethanol treatment, which, respectively, inhibits or prevents activation of caspase 3, rescued the DNMT1 and DNMT3A proteins and DNA methylation. No reduction of DNMT1 and DNMT3A proteins and DNA methylation was found in P7 CB1R null mice, which exhibit no ethanol‐induced activation of caspase 3. Together, these data demonstrate that ethanol‐induced activation of caspase 3 impairs DNA methylation through DNMT1 and DNMT3A in the neonatal mouse brain, and such impairments are absent in CB1R null mice. Epigenetic events mediated by DNA methylation may be one of the essential mechanisms of ethanol teratogenesis.

     

Shoring up DNA methylation and H3K27me3 domain demarcation at developmental genes

Mutual antagonism between DNA methylation and H3K27me3 histone methylation suggests a dynamic crosstalk between these epigenetic marks that could help ensure correct gene expression programmes. Work from Manzo et al ( 2017 ) now shows that an isoform of de novo DNA methyltransferase DNMT3A provides specificity in the system by depositing DNA methylation at adjacent “shores” of hypomethylated bivalent CpG islands (CGI) in mouse embryonic stem cells (mESCs). DNMT3A1‐directed methylation appears to be instructive in maintaining the H3K27me3 profile at the hypomethylated bivalent CGI promoters of developmentally important genes.     

RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

Site-specific hypermethylation of tumor suppressor genes accompanied by genome-wide hypomethylation are epigenetic hallmarks of malignancy. However, the molecular mechanisms that drive these linked changes in DNA methylation remain obscure. DNA methyltransferase 1 (DNMT1), the principle enzyme responsible for maintaining methylation patterns is commonly dysregulated in tumors. Replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in a gain of DNMT1 function. However, a substantial body of data suggested that RFTS is required for DNMT1 activity. Here, we demonstrate that deletion of RFTS alters DNMT1-dependent DNA methylation during malignant transformation. Compared to full-length DNMT1, ectopic expression of hyperactive DNMT1-ΔRFTS caused greater malignant transformation and enhanced promoter methylation with condensed chromatin structure that silenced DAPK and DUOX1 expression. Simultaneously, deletion of RFTS impaired DNMT1 chromatin association with pericentromeric Satellite 2 (SAT2) repeat sequences and produced DNA demethylation at SAT2 repeats and globally. To our knowledge, RFTS-deleted DNMT1 is the first single factor that can reprogram focal hypermethylation and global hypomethylation in parallel during malignant transformation. Our evidence suggests that the RFTS domain of DNMT1 is a target responsible for epigenetic changes in cancer.     

Aberrant signature methylome by DNMT1 hot spot mutation in hereditary sensory and autonomic neuropathy 1E

DNA methyltransferase 1 (DNMT1) is essential for DNA methylation, gene regulation and chromatin stability. We previously discovered DNMT1 mutations cause hereditary sensory and autonomic neuropathy type 1 with dementia and hearing loss (HSAN1E; OMIM 614116). HSAN1E is the first adult-onset neurodegenerative disorder caused by a defect in a methyltransferase gene. HSAN1E patients appear clinically normal until young adulthood, then begin developing the characteristic symptoms involving central and peripheral nervous systems. Some HSAN1E patients also develop narcolepsy and it has recently been suggested that HSAN1E is allelic to autosomal dominant cerebellar ataxia, deafness, with narcolepsy (ADCA-DN; OMIM 604121), which is also caused by mutations in DNMT1. A hotspot mutation Y495C within the targeting sequence domain of DNMT1 has been identified among HSAN1E patients. The mutant DNMT1 protein shows premature degradation and reduced DNA methyltransferase activity. Herein, we investigate genome-wide DNA methylation at single-base resolution through whole-genome bisulfite sequencing of germline DNA in 3 pairs of HSAN1E patients and their gender- and age-matched siblings. Over 1 billion 75-bp single-end reads were generated for each sample. In the 3 affected siblings, overall methylation loss was consistently found in all chromosomes with X and 18 being most affected. Paired sample analysis identified 564,218 differentially methylated CpG sites (DMCs; P < 0.05), of which 300 134 were intergenic and 264 084 genic CpGs. Hypomethylation was predominant in both genic and intergenic regions, including promoters, exons, most CpG islands, L1, L2, Alu, and satellite repeats and simple repeat sequences. In some CpG islands, hypermethylated CpGs outnumbered hypomethylated CpGs. In 201 imprinted genes, there were more DMCs than in non-imprinted genes and most were hypomethylated. Differentially methylated region (DMR) analysis identified 5649 hypomethylated and 1872 hypermethylated regions. Importantly, pathway analysis revealed 1693 genes associated with the identified DMRs were highly associated in diverse neurological disorders and NAD+/NADH metabolism pathways is implicated in the pathogenesis. Our results provide novel insights into the epigenetic mechanism of neurodegeneration arising from a hotspot DNMT1 mutation and reveal pathways potentially important in a broad category of neurological and psychological disorders.