DNA methylation (5-methylcytosine, 5mC) is a major form of DNA modification in the mammalian genome that plays critical roles in chromatin structure and gene expression. In general, DNA methylation ...is stably maintained in somatic tissues. However, DNA methylation patterns and levels show dynamic changes during development. Specifically, the genome undergoes two waves of global demethylation and remethylation for the purpose of producing the next generation. The first wave occurs in the germline, initiated with the erasure of global methylation in primordial germ cells (PGCs) and completed with the establishment of sex-specific methylation patterns during later stages of germ cell development. The second wave occurs after fertilization, including the erasure of most methylation marks inherited from the gametes and the subsequent establishment of the embryonic methylation pattern. The two waves of DNA methylation reprogramming involve both distinct and shared mechanisms. In this review article, we provide an overview of the key reprogramming events, focusing on the important players in these processes, including DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) family of 5mC dioxygenases.
Cellular differentiation is, by definition, epigenetic. Genome-wide profiling of pluripotent cells and differentiated cells suggests global chromatin remodelling during differentiation, which results ...in a progressive transition from a fairly open chromatin configuration to a more compact state. Genetic studies in mouse models show major roles for a variety of histone modifiers and chromatin remodellers in key developmental transitions, such as the segregation of embryonic and extra-embryonic lineages in blastocyst stage embryos, the formation of the three germ layers during gastrulation and the differentiation of adult stem cells. Furthermore, rather than merely stabilizing the gene expression changes that are driven by developmental transcription factors, there is emerging evidence that chromatin regulators have multifaceted roles in cell fate decisions.
DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA ...interference pathway for transcriptional gene silencing （TGS）, which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages： during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair （BER） pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review.
DNA methylation plays a critical role in the regulation of chromatin structure and gene expression and is involved in a variety of biological processes. The levels and patterns of DNA methylation are ...regulated by both DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and 'demethylating' proteins, including the ten-eleven translocation (TET) family of dioxygenases (TET1, TET2 and TET3). The effects of DNA methylation on chromatin and gene expression are largely mediated by methylated DNA 'reader' proteins, including MeCP2. Numerous mutations in
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have been identified in cancer and developmental disorders, highlighting the importance of the DNA methylation machinery in human development and physiology. In this review, we describe these mutations and discuss how they may lead to disease phenotypes.
Methylation of cytosines is a major epigenetic modification in mammalian genomes. The levels and patterns of DNA methylation are the results of the opposing actions of methylating and demethylating ...machineries. Over the past two decades, great progress has been made in elucidating the methylating machinery including the identification and functional characterization of the DNA methyltransferases (Dnmts). However, the mechanisms of demethylation and the major players involved had been elusive. A major breakthrough came in 2009, when the ten-eleven translocation (Tet) family of proteins was discovered as 5-methylcytosine (5mC) dioxygenases that convert 5mC to 5-hydroxymethylcytosine (5hmC). Studies in the past several years have established that 5hmC serves as an intermediate in DNA demethylation and that Tet proteins have important roles in epigenetic reprogramming in early embryos and primordial germ cells. In this review, we discuss recent advances in this exciting field, focusing on the role of Tet proteins in mammalian development.
The de novo DNA methyltransferases Dnmt3a and Dnmt3b play crucial roles in developmental and cellular processes. Their enzymatic activities are stimulated by a regulatory protein Dnmt3L (Dnmt3-like) ...in vitro. However, genetic evidence indicates that Dnmt3L functions predominantly as a regulator of Dnmt3a in germ cells. How Dnmt3a and Dnmt3b activities are regulated during embryonic development and in somatic cells remains largely unknown. Here we show that Dnmt3b3, a catalytically inactive Dnmt3b isoform expressed in differentiated cells, positively regulates de novo methylation by Dnmt3a and Dnmt3b with a preference for Dnmt3b. Dnmt3b3 is equally potent as Dnmt3L in stimulating the activities of Dnmt3a2 and Dnmt3b2 in vitro. Like Dnmt3L, Dnmt3b3 forms a complex with Dnmt3a2 with a stoichiometry of 2:2. However, rescue experiments in
triple-knockout (TKO) mouse embryonic stem cells (mESCs) reveal that Dnmt3b3 prefers Dnmt3b2 over Dnmt3a2 in remethylating genomic sequences. Dnmt3a2, an active isoform that lacks the N-terminal uncharacterized region of Dnmt3a1 including a nuclear localization signal, has very low activity in TKO mESCs, indicating that an accessory protein is absolutely required for its function. Our results suggest that Dnmt3b3 and perhaps similar Dnmt3b isoforms facilitate de novo DNA methylation during embryonic development and in somatic cells.
DNA methylation is a heritable epigenetic mark, enabling stable but reversible gene repression. In mammalian cells, DNA methyltransferases (DNMTs) are responsible for modifying cytosine to ...5-methylcytosine (5mC), which can be further oxidized by the TET dioxygenases to ultimately cause DNA demethylation. However, the genome-wide cooperation and functions of these two families of proteins, especially at large under-methylated regions, called canyons, remain largely unknown.
Here we demonstrate that DNMT3A and TET1 function in a complementary and competitive manner in mouse embryonic stem cells to mediate proper epigenetic landscapes and gene expression. The longer isoform of DNMT3A, DNMT3A1, exhibits significant enrichment at distal promoters and canyon edges, but is excluded from proximal promoters and canyons where TET1 shows prominent binding. Deletion of Tet1 increases DNMT3A1 binding capacity at and around genes with wild-type TET1 binding. However, deletion of Dnmt3a has a minor effect on TET1 binding on chromatin, indicating that TET1 may limit DNA methylation partially by protecting its targets from DNMT3A and establishing boundaries for DNA methylation. Local CpG density may determine their complementary binding patterns and therefore that the methylation landscape is encoded in the DNA sequence. Furthermore, DNMT3A and TET1 impact histone modifications which in turn regulate gene expression. In particular, they regulate Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 enrichment to constrain gene expression from bivalent promoters.
We conclude that DNMT3A and TET1 regulate the epigenome and gene expression at specific targets via their functional interplay.
Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation ...establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.
Proper telomere length is essential for embryonic stem cell (ESC) self-renewal and pluripotency. Mouse ESCs (mESCs) sporadically convert to a transient totipotent state similar to that of two-cell ...(2C) embryos to recover shortened telomeres. Zscan4, which exhibits a burst of expression in 2C-like mESCs, is required for telomere extension in these cells. However, the mechanism by which Zscan4 extends telomeres remains elusive. Here, we show that Zscan4 facilitates telomere elongation by inducing global DNA demethylation through downregulation of Uhrf1 and Dnmt1, major components of the maintenance DNA methylation machinery. Mechanistically, Zscan4 recruits Uhrf1 and Dnmt1 and promotes their degradation, which depends on the E3 ubiquitin ligase activity of Uhrf1. Blocking DNA demethylation prevents telomere elongation associated with Zscan4 expression, suggesting that DNA demethylation mediates the effect of Zscan4. Our results define a molecular pathway that contributes to the maintenance of telomere length homeostasis in mESCs.
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•2C-like mESCs show global DNA hypomethylation•Zscan4 is responsible for DNA demethylation in 2C-like mESCs•Zscan4 induces Uhrf1-dependent ubiquitination and degradation of Uhrf1 and Dnmt1•Blocking DNA demethylation prevents Zscan4-mediated telomere elongation
Mouse embryonic stem cells sporadically convert to a transient totipotent (2C-like) state in which shortened telomeres are extended dependent on Zscan4. Dan et al. demonstrate that Zscan4 facilitates telomere elongation by inducing Uhrf1-dependent Uhrf1 and Dnmt1 degradation, leading to global DNA demethylation.
Somatic heterozygous mutations of the DNA methyltransferase gene DNMT3A occur frequently in acute myeloid leukemia and other hematological malignancies, with the majority (∼60%) of mutations ...affecting a single amino acid, Arg882 (R882), in the catalytic domain. Although the mutations impair DNMT3A catalytic activity in vitro, their effects on DNA methylation in cells have not been explored. Here, we show that exogenously expressed mouse Dnmt3a proteins harboring the corresponding R878 mutations largely fail to mediate DNA methylation in murine embryonic stem (ES) cells but are capable of interacting with wild-type Dnmt3a and Dnmt3b. Coexpression of the Dnmt3a R878H (histidine) mutant protein results in inhibition of the ability of wild-type Dnmt3a and Dnmt3b to methylate DNA in murine ES cells. Furthermore, expression of Dnmt3a R878H in ES cells containing endogenous Dnmt3a or Dnmt3b induces hypomethylation. These results suggest that the DNMT3A R882 mutations, in addition to being hypomorphic, have dominant-negative effects.
•Mouse Dnmt3a R878H (human R882H) mutant protein inhibits wild-type Dnmt3a/Dnmt3b in murine ES cells, suggesting dominant-negative effects.
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