Pericentric Heterochromatin Generated by HP1 Protein Interaction-defective Histone Methyltransferase Suv39h1

Pericentric regions form epigenetically organized silent heterochromatin structures that accumulate histone H3 lysine 9 trimethylation (H3K9me3) and HP1. At pericentric regions, Suv39h is the major enzyme that generates H3K9me3. Suv39h also interacts directly with HP1, a methylated H3K9-binding protein. However, it is not well characterized how HP1 interaction is important for Suv39h accumulation and Suv39h-mediated H3K9me3 formation at the pericentromere. To address this, we introduced the HP1 binding-defective N-terminally truncated mouse Suv39h1 (ΔN) into Suv39h-deficient embryonic stem cells. Interestingly, pericentric accumulation of ΔN and ΔN-mediated H3K9me3 was observed to recover, but HP1 accumulation was only marginally restored. ΔN also rescued DNA methyltransferase Dnmt3a and -3b accumulation and DNA methylation of the pericentromere. In contrast, other pericentric heterochromatin features, such as ATRX protein association and H4K20me3, were not recovered. Finally, derepressed major satellite repeats were partially silenced by ΔN expression. These findings clearly showed that the Suv39h-HP1 binding is dispensable for pericentric H3K9me3 and DNA methylation, but this interaction and HP1 recruitment/accumulation seem to be crucial for complete formation of heterochromatin.     

A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells

The Rb/E2F complex represses S-phase genes both in cycling cells and in cells that have permanently exited from the cell cycle and entered a terminal differentiation pathway. Here we show that S-phase gene repression, which involves histone-modifying enzymes, occurs through distinct mechanisms in these two situations. We used chromatin immunoprecipitation to show that methylation of histone H3 lysine 9 (H3K9) occurs at several Rb/E2F target promoters in differentiating cells but not in cycling cells. Furthermore, phenotypic knock-down experiments using siRNAs showed that the histone methyltransferase Suv39h is required for histone H3K9 methylation and subsequent repression of S-phase gene promoters in differentiating cells, but not in cycling cells. These results indicate that the E2F target gene permanent silencing mechanism that is triggered upon terminal differentiation is distinct from the transient repression mechanism in cycling cells. Finally, Suv39h-depleted myoblasts were unable to express early or late muscle differentiation markers. Thus, appropriately timed H3K9 methylation by Suv39h seems to be part of the control switch for exiting the cell cycle and entering differentiation.     

The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase

The DNA methyltransferases, Dnmts, are the enzymes responsible for methylating DNA in mammals, which leads to gene silencing. Repression by DNA methylation is mediated partly by recruitment of the methyl-CpG-binding protein MeCP2. Recently, MeCP2 was shown to associate and facilitate histone methylation at Lys9 of H3, which is a key epigenetic modification involved in gene silencing. Here, we show that endogenous Dnmt3a associates primarily with histone H3-K9 methyltransferase activity as well as, to a lesser extent, with H3-K4 enzymatic activity. The association with enzymatic activity is mediated by the conserved PHD-like motif of Dnmt3a. The H3-K9 histone methyltransferase that binds Dnmt3a is likely the H3-K9 specific SUV39H1 enzyme since we find that it interacts both in vitro and in vivo with Dnmt3a, using its PHD-like motif. We find that SUV39H1 also binds to Dnmt1 and, consistent with these interactions, SUV39H1 can purify DNA methyltransferase activity from nuclear extracts. In addition, we show that HP1β, a SUV39H1-interacting partner, binds directly to Dnmt1 and Dnmt3a and that native HP1β associates with DNA methyltransferase activity. Our data show a direct connection between the enzymes responsible for DNA methylation and histone methylation. These results further substantiate the notion of a self-reinforcing repressive chromatin state through the interplay between these two global epigenetic modifications.     

Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells

It has been reported that DNA methyltransferase 1-deficient (Dnmt1-/-) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a-/-,3b-/-] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a-/-,3b-/-] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a-/-,3b-/-] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1-/- ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1.     

DNA hypermethylation in Drosophila melanogaster causes irregular chromosome condensation and dysregulation of epigenetic histone modifications

The level of genomic DNA methylation plays an important role in development and disease. In order to establish an experimental system for the functional analysis of genome-wide hypermethylation, we overexpressed the mouse de novo methyltransferase Dnmt3a in Drosophila melanogaster. These flies showed severe developmental defects that could be linked to reduced rates of cell cycle progression and irregular chromosome condensation. In addition, hypermethylated chromosomes revealed elevated rates of histone H3-K9 methylation and a more restricted pattern of H3-S10 phosphorylation. The developmental and chromosomal defects induced by DNA hypermethylation could be rescued by mutant alleles of the histone H3-K9 methyltransferase gene Su(var)3-9. This mutation also resulted in a significantly decreased level of genomic DNA methylation. Our results thus uncover the molecular consequences of genomic hypermethylation and demonstrate a mutual interaction between DNA methylation and histone methylation.     

The HP1α–CAF1–SetDB1‐containing complex provides H3K9me1 for Suv39‐mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

EBP1 regulates Suv39H1 stability via the ubiquitin-proteasome system in neural development

ErbB3-binding protein 1 (EBP1) is a multifunctional protein associated with neural development. Loss of Ebp1 leads to upregulation of the gene silencing unit suppressor of variegation 3-9 homolog 1 (Suv39H1)/DNA (cytosine 5)-methyltransferase (DNMT1). EBP1 directly binds to the promoter region of DNMT1, repressing DNA methylation, and hence, promoting neural development. In the current study, we showed that EBP1 suppresses histone methyltransferase activity of Suv39H1 by promoting ubiquitin-proteasome system (UPS)-dependent degradation of Suv39H1. In addition, we showed that EBP1 directly interacts with Suv39H1, and this interaction is required for recruiting the E3 ligase MDM2 for Suv39H1 degradation. Thus, our findings suggest that EBP1 regulates UPS-dependent degradation of Suv39H1 to govern proper heterochromatin assembly during neural development.     

Histone tails regulate DNA methylation by allosterically activating de novo methyltransferase

Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyltransferase is largely unknown. Here, we show that histone H3 tails lacking lysine 4 (K4) methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain (PHD). In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.     

DNA甲基化与脊椎动物胚胎发育

DNA甲基化是指DNA甲基转移酶(DNMT)将DNA序列中的5′胞嘧啶转变为5′甲基胞嘧啶的化学修饰, 可以调控基因的时空特异性表达, 从而影响细胞命运决定和分化等生物学过程。近年来研究发现, DNA甲基化在脊椎动物胚胎早期发育中有重要作用, Dnmt基因的缺失会影响胚胎早期发育和多个器官的形成及分化, 如胚胎早期致死、内脏器官和神经系统终末分化缺陷以及血液发生紊乱等。文章总结了DNA甲基化转移酶在小鼠和斑马鱼发育过程中的动态变化, 并系统阐述了DNA甲基化在胚胎早期发育和器官发生中的作用, 重点揭示DNA 甲基化转移酶与组蛋白甲基化转移酶如何协同调控DNA甲基化从而影响基因转录的分子机理。DNA甲基化作为一种关键的表观遗传学因素, 全面系统地理解其在胚胎发育过程中的作用机制对靶向治疗人类相关疾病有一定的理论指导意义。     

Replication-independent chromatin loading of Dnmt1 during G2 and M phases

The major DNA methyltransferase, Dnmt1, associates with DNA replication sites in S phase maintaining the methylation pattern in the newly synthesized strand. In view of the slow kinetics of Dnmt1 in vitro versus the fast progression of the replication fork, we have tested whether Dnmt1 associates with chromatin beyond S phase. Using time-lapse microscopy of mammalian cells expressing green-fluorescent-protein-tagged Dnmt1 and DsRed-tagged DNA Ligase I as a cell cycle progression marker, we have found that Dnmt1 associates with chromatin during G2 and M. This association is mediated by a specific targeting sequence, shows strong preference for constitutive but not facultative heterochromatin and is independent of heterochromatin-specific histone H3 Lys 9 trimethylation, SUV39H and HP1. Moreover, photobleaching analyses showed that Dnmt1 is continuously loaded onto chromatin throughout G2 and M, indicating a replication-independent role of Dnmt1 that could represent a novel and separate pathway to maintain DNA methylation.     

DNA methyltransferase 3b preferentially associates with condensed chromatin

In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a 'reader' of higher-order chromatin structure leading to gene silencing through DNA methylation.