Differential acetylation of histone H4 lysine during development of in vitro fertilized,cloned and parthenogenetically activated bovine embryos

The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterise the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense. In IVF zygotes, both pronuclei were transiently hyper-acetylated. However, the male pronucleus was faster in acquiring acetylated histones, and concurrently it was rapidly demethylated. Both pronuclei were equally acetylated during the S to G2-phase transition, while methylation staining was only still observed in the female pronucleus. In parthenogenetically activated oocytes, acetylation of the female pronucleus was enriched faster, while DNA remained methylated. A transient de-acetylation was observed in NT embryos reconstructed using a non-activated ooplast of a metaphase second arrested oocyte. Remarkably, the intensity of acetylation staining of most H4 lysine residues peaked at the 8-cell stage in IVF embryos, which coincided with zygotic genome activation and with lowest DNA methylation staining. At the blastocyst stage, trophectodermal cells of IVF and parthenogenetic embryos generally demonstrated more intense staining for most acetylated H4 lysine, whilst ICM cells stained very weakly. In contrast methylation of the DNA stained more intensely in ICM. NT blastocysts showed differential acetylation of blastomeres but not methylation. The inverse association of histone lysine acetylation and DNA methylation at different vital embryo stages suggests a mechanistically significant relationship. The complexities of these epigenetic interactions are discussed.     

DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium

Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation, a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2' deoxycytidine during acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19, a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which mammary epithelial differentiation is coordinated both at the tissue and cellular levels.     

Trichostatin A decreases the levels of MeCP2 expression and phosphorylation and increases its chromatin binding affinity

MeCP2 binds to methylated DNA in a chromatin context and has an important role in cancer and brain development and function. Histone deacetylase (HDAC) inhibitors are currently being used to palliate many cancer and neurological disorders. Yet, the molecular mechanisms involved are not well known for the most part and, in particular, the relationship between histone acetylation and MeCP2 is not well understood. In this paper, we study the effect of the HDAC inhibitor trichostatin A (TSA) on MeCP2, a protein whose dysregulation plays an important role in these diseases. We find that treatment of cells with TSA decreases the phosphorylation state of this protein and appears to result in a higher MeCP2 chromatin binding affinity. Yet, the binding dynamics with which the protein binds to DNA appear not to be significantly affected despite the chromatin reorganization resulting from the high levels of acetylation. HDAC inhibition also results in an overall decrease in MeCP2 levels of different cell lines. Moreover, we show that miR132 increases upon TSA treatment, and is one of the players involved in the observed downregulation of MeCP2.     

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.     

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.     

Ellagic acid inhibits adipocyte differentiation through coactivator-associated arginine methyltransferase 1-mediated chromatin modification

Chromatin remodeling is a key mechanism in adipocyte differentiation. However, it is unknown whether dietary polyphenols are epigenetic effectors for adiposity control. Ellagic acid (EA) is a naturally occurring polyphenol in numerous fruits and vegetables. Recently, EA-containing foods have been reported to reduce adiposity. In the present study, we sought to determine whether EA inhibits adipogenesis by modifying chromatin remodeling in human adipogenic stem cells (hASCs). qPCR microarray of chromatin modification enzymes revealed that 10 μmol/L of EA significantly inhibits histone deacetylase (HDAC)9 down-regulation. In addition, EA was associated with up-regulation of HDAC activity and a marked reduction of histone acetylation levels. However, chemical inhibition of HDAC activity or depletion of HDAC9 by siRNA were not sufficient to reverse the antiadipogenic effects of EA. Intriguingly, EA treatment was also associated with reduced histone 3 arginine 17 methylation levels (H3R17me2), implying the inhibitory role of EA in coactivator-associated arginine methyltransferase 1 (CARM)1 activity during adipogenesis. Boosting CARM1 activity by delivering cell-penetrating peptides of CARM1 not only recovered H3R17me2 but also restored adipogenesis evidenced by H3 acetylation at lysine 9, HDAC9 down-regulation, PPARγ expression and triglyceride accumulation. Taken together, our data suggest that reduced CARM1 activity by EA results in a decrease of H3R17me2 levels, which may interrupt consecutive histone remodeling steps for adipocyte differentiation including histone acetylation and HDAC9 dissociation from chromatin. Our work provides the mechanistic insights into how EA, a polyphenol ubiquitously found in fruits and vegetables, attenuates human adipocyte differentiation by altering chromatin remodeling.     

Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes

In different eukaryotic model systems, chromatin and gene expression are modulated by post-translational modification of histone tails. In this in vivo study, histone methylation and acetylation are investigated along the imprinted mouse genes Snrpn, Igf2r and U2af1-rs1. These imprinted genes all have a CpG-rich regulatory element at which methylation is present on the maternal allele, and originates from the female germ line. At these 'differentially methylated regions' (DMRs), histone H3 on the paternal allele has lysine-4 methylation and is acetylated. On the maternally inherited allele, in contrast, chromatin is marked by hypermethylation on lysine-9 of H3. Allele-specific patterns of lysine-4 and lysine-9 methylation are also detected at other regions of the imprinted loci. For the DMR at the U2af1-rs1 gene, we establish that the methyl-CpG-binding-domain (MBD) proteins MeCP2, MBD1 and MBD3 are associated with the maternal allele. These data support the hypothesis that MBD protein-associated histone deacetylase/chromatin-remodelling complexes are recruited to the parental allele that has methylated DNA and H3-K9 methylation, and are prevented from binding to the opposite allele by H3 lysine-4 methylation.     

MeCP2 preferentially binds to methylated linker DNA in the absence of the terminal tail of histone H3 and independently of histone acetylation

Methyl CpG binding protein 2 (MeCP2) is a basic protein that contains a DNA methyl binding domain. The mechanism by which the highly positive charge of MeCP2 and its ability to bind methylated DNA contribute to the specificity of its binding to chromatin has long remained elusive. In this paper, we show that MeCP2 binds to nucleosomes in a very similar way to linker histones both in vitro and in vivo. However, its binding specificity strongly depends on DNA methylation. We also observed that as with linker histones, this binding is independent of the core histone H3 N-terminal tail and is not affected by histone acetylation.     

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.     

Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx

Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β cells was bound by methyl-binding protein MeCP2, which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain pancreatic β cell identity.