DNA methylation plays an important role in mammalian development and correlates with chromatinassociated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys 9 of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys 9 H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys 9 methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.Methylation of cytosines is essential for mammalian development and is associated with gene silencing (1). DNA methylation represses genes partly by recruitment of methyl-CpGbinding domain proteins, which selectively recognize methylated CpG dinucleotides. MeCP2 is the founder member of the methyl-CpG-binding domain proteins, which consists of a single polypeptide that contains a methyl-CpG-binding domain and a transcriptional repression domain. MeCP2 is capable of binding to a single symmetrically methylated CpG both in naked DNA and within chromatin (2, 3).It is now well established that MeCP2 silences transcription by recruiting the histone deacetylase (HDAC) 1 repressive machinery, which removes acetyl groups from histones resulting in gene silencing (4,5). However, the inhibition of histone deacetylase activity using drugs such as Trichostatin A only partially relieves MeCP2-mediated transcriptional repression. This partial relief indicates that additional mechanisms of repression by MeCP2 likely exist aside from the recruitment of histone deacetylase.Beside histone deacetylation, histone methylation is emerging as another key post-translational modification of histones and represents an important epigenetic mechanism for the organization of chromatin structure and the regulation of gene expression. In particular, methylation at lysine 9 of histone H3 is associated with gene silencing, and several enzymes that catalyze the addition of methyl groups to lysine 9 have recently been identified (6). Interestingly, recent data have shown that the retinoblastoma protein represses transcription through the recruitment of HDAC activity, but in a second step it recruits histone methylation activity specific for lysine 9 of histone H3 (7). By analogy to retinoblastoma, we considered in the present work whether MeCP2-mediated repression might also include, besides histone deacetylation, a second stage involving histone methylatio...

Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.Hha I) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.Hha I and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine.

Upon fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA methylation and histone modifications. Currently, it is not known whether replacement of the major histones by histone variants is also involved in these processes. Here we have examined the expression and localization of the histone variant H3.3 in early mouse embryogenesis. We show that H3.3 is present in the oocyte as a maternal factor. It is then incorporated preferentially into the male pronucleus before genome activation, pointing towards an asymmetry in histone composition between the two pronuclei. This is in line with the male pronucleus bearing transcriptional activation first. The same distribution was observed when we followed the localisation of a tagged version of H3.3. We detected H3.3 in the nuclei of mouse embryos in all of the stages analysed, from the zygote to the blastocyst stage, suggesting that the epigenetic mechanisms in the early embryo not only involve changes in histone modifications but may also include histone replacement.

Several recent studies from the field of epigenetics have combined chromatin-immunoprecipitation (ChIP) with next-generation high-throughput sequencing technologies to describe the locations of histone post-translational modifications (PTM) and DNA methylation genome-wide. While these reports begin to quench the chromatin biologists thirst for visualizing where in the genome epigenetic marks are placed, they also illustrate several advantages of sequencing based genomics compared to microarray analysis. Accordingly, next-generation sequencing (NGS) technologies are now challenging microarrays as the tool of choice for genome analysis. The increased affordability of comprehensive sequence-based genomic analysis will enable new questions to be addressed in many areas of biology. It is inevitable that massively-parallel sequencing platforms will supercede the microarray for many applications, however, there are niches for microarrays to fill and interestingly we may very well witness a symbiotic relationship between microarrays and high-throughput sequencing in the future.