Showing papers by "Yoichi Shinkai published in 2007"

Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice.

Abstract: We previously reported that primordial germ cells (PGCs) in mice erase genome-wide DNA methylation and histone H3 lysine9 dimethylation (H3K9me2), and instead acquire high levels of tri-methylation of H3K27 (H3K27me3) during their migration, a process that might be crucial for the re-establishment of potential totipotency in the germline. We here explored a cellular dynamics associated with this epigenetic reprogramming. We found that PGCs undergo erasure of H3K9me2 and upregulation of H3K27me3 in a progressive, cell-by-cell manner, presumably depending on their developmental maturation. Before or concomitant with the onset of H3K9 demethylation, PGCs entered the G2 arrest of the cell cycle, which apparently persisted until they acquired high H3K27me3 levels. Interestingly, PGCs exhibited repression of RNA polymerase II-dependent transcription, which began after the onset of H3K9me2 reduction in the G2 phase and tapered off after the acquisition of high-level H3K27me3. The epigenetic reprogramming and transcriptional quiescence were independent from the function of Nanos3. We found that before H3K9 demethylation, PGCs exclusively repress an essential histone methyltransferase, GLP, without specifically upregulating histone demethylases. We suggest the possibility that active repression of an essential enzyme and subsequent unique cellular dynamics ensures successful implementation of genome-wide epigenetic reprogramming in migrating PGCs.

Functional dynamics of H3K9 methylation during meiotic prophase progression

Abstract: Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. G9a is a major mammalian H3K9 methyltransferase at euchromatin and is essential for mouse embryogenesis. Here we describe the roles of G9a in germ cell development. Mutant mice in which G9a is specifically inactivated in the germ-lineage displayed sterility due to a drastic loss of mature gametes. G9a-deficient germ cells exhibited perturbation of synchronous synapsis in meiotic prophase. Importantly, mono- and di-methylation of H3K9 (H3K9me1 and 2) in G9a-deficient germ cells were significantly reduced and G9a-regulated genes were overexpressed during meiosis, suggesting that G9a-mediated epigenetic gene silencing is crucial for proper meiotic prophase progression. Finally, we show that H3K9me1 and 2 are dynamically and sex-differentially regulated during the meiotic prophase. This genetic and biochemical evidence strongly suggests that a specific set of H3K9 methyltransferase(s) and demethylase(s) coordinately regulate gametogenesis.

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells

Abstract: In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a−/− embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a−/− cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a−/− cells. These data indicate that G9a site-selectively contributes to DNA methylation.

Regulation and function of H3K9 methylation.

Abstract: First histone lysine methyltransferase (HLMTase) was discovered in 2000. Since then, there are reports of dozens of novel HLMTases in different eukaryotes including plant, fungus, insect, nematode and vertebrate. The enzymes and their specific histone-lysine modifications have enormous impacts on the regulation of chromatin structure and function. Furthermore, various histone methyl-lysine demethylases (HLDMases) have been identified recently. In this chapter, histone H3 lysine 9 specific methyltransferases will be discussed as model enzymes involved in the regulation of chromatin function

Identification of ZNF200 as a novel binding partner of histone H3 methyltransferase G9a.

Abstract: G9a belongs to the subfamily of histone H3 lysine 9 (H3-K9)-specific methyltransferases. On amino acid sequence alignment of human and Drosophila G9a, we found that the N-terminal region from amino acids 532–605 to be evolutionarily conserved and named this the G9a homology domain (GHD). Using the GHD of human G9a (hG9a) as a bait, we isolated cDNA encoding a zinc finger protein 200 (ZNF200), which contains five C2H2-type zinc finger domains in tandem arrays. Interaction between G9a and ZNF200 could be demonstrated by in vitro binding assays and immunoprecipitation experiments using cultured human HEK293 cell extracts. GST pull-down assays using deletion derivatives of ZNF200 revealed that the interaction is through a region encompassing three of the five zinc finger domains. Furthermore, ZNF200 appear to co-localize with G9a in the nucleoplasm of HEK293 cells as discrete speckles. These results demonstrate that ZNF200 is a novel binding partner of G9a.