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Laboratory for Developmental Genetics Group director Haruhiko Koseki Researchers Technical Staffs Student Trainee Visiting Scientist : Mitsuhiro Endo, Yûichi Fujimura, Shûichi Hiraoka, Kyôichi Isono, Yuki Takada, Jun Shinga : Takanori Hasegawa, Midori Iida, Iyo Kataoka, Naoko Ohnaga, Hiroshi Kajita, Yasuaki Murahashi, Shinobu Mochizuki, Masashi Matsuda, Tomoyuki Ishikura, Tamie Morisawa, Atushi Kajiwara, Yôko Koseki, Rie Suzuki, Kayoko Katsuyama, Naomi Ootsuka, Isamu Hisanaga, Momoko Ogoshi, Natsumi Saito, : Masayo Harada (JRA) : Mutô Masahiro The Developmental Genetics Research Group fulfills a double role within RCAI. A large portion of the manpower and financial resources of the group is devoted to the maintenance of a high-standard mouse facility at RCAI. Through the Animal Core Facility, the group is also responsible for the generation of knock-out and transgenic animals for the various research laboratories at the center. At the same time, the laboratory is pursuing a research program to elucidate the molecular mechanisms underlying the epigenetic regulation mediated by Polycomb group (PcG) proteins in development. PcG genes were first identified in Drosophila melanogaster as a cluster of genes required for maintenance of segmental identity and were subsequently shown to be structurally and functionally conserved in mammals. Genetic analyses of mammalian PcG proteins revealed their roles not only in anterior-posterior specification but also in cellular proliferation, differentiation, and senescence. In particular, significant impact of PcG functions on lymphocyte and lymphoid organ development have frequently been reported. PcG gene products form at least two different multimeric protein complexes; one mediates histone H3-K27 trimethylation and the other H2A-K119 ubiquitinylation on the chromatin. It is, however, unknown how PcG complexes mediate transcriptional repression and, consequently, exert their biological functions. To elucidate the molecular nature of PcG-mediated transcriptional regulation, we have been focusing on the following issues; (1) regulatory mechanisms of PcG binding to their targets, and (2) regulation of PcG functions by interacting proteins. Regulatory mechanisms of PcG binding to their targets (Endoh et al., Submitted) To address the molecular mechanisms underlying PcG-mediated heritable silencing of developmental regulators, we have focused on the role of these proteins in embryonic stem (ES) cells. The Polycomb repressive complexes-2 (PRC2) has been shown to share target genes with the core transcription network, including Oct3/4, Sox2 and Nanog, to maintain ES cells in an undifferentiated state. However, it is still unclear whether and how PcG and the core transcription network are functionally linked. We have identified an essential role for the core components of Polycomb repressive complexes-1 (PRC1), Ring1A and Ring1B, in Assistants : Ryôko Moriizumi, Hiroko Iwamoto 24
Recent publications DAPI dig-dUTP Np95 Merge TKO+5me-dCTP Sharif J., Muto M., Takebayashi S., Suetake I., Iwamatsu A., Endo T.A., Shinga J., Mizutani-Koseki Y., Toyoda T., Okamura K., Tajima S., Mitsuya K., Okano M., Koseki H. (2007) The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450:908-912. Figure Recognition of hemimethylated DNA by Np95. Dnmt1/3a/3b triple knockout (TKO) ES cells were labeled by either methylated dCTP (TKO+ 5me - dCTP) or unmethylated (TKO+ dCTP) tother with dig-dUTP. Note the accumulation of Np95 to newly replicated heterochromatic regions (marked by colocalization of DAPI and dig-dUTP) in TKO cells that incorporated methylated dCTP. repressing developmental regulators in ES cells and thereby in maintaining ES cell identity. A significant proportion of the PRC1 target genes are also repressed by Oct3/4. We could show that engagement of PRC1 at target genes is Oct3/4-dependent whereas engagement of Oct3/4 is PRC1 independent. Moreover, upon differentiation induced by Gata6 expression, most of the Ring1A/B target genes are derepressed and the binding of Ring1A/B to their target loci is also decreased. Collectively these results have allowed us to conclude that Ring1A/B-mediated Polycomb silencing functions downstream of the core transcriptional regulatory circuitry to maintain ES cell identity. Regulation of PcG functions by interacting proteins (Sharif et al., Nature) We identified a SET and RING fingerassociated (SRA) domain protein Np95 (also called UHRF1 or ICBP90) as an interacting protein for Mel18, a PRC1 component. Purification of Np95 complexes revealed its association with not only PRC1 components but also with DNA (cytosine-5-)-methyltransferase 1 (Dnmt1), which is the principal enzyme responsible for maintenance of CpG methylation and is essential for the regulation of gene expression, silencing of parasitic DNA elements, TKO+dCTP genomic imprinting, and embryogenesis. This year, we focused on the functional link between Np95 and Dnmt1. Dnmt1 is needed in S-phase to methylate newly replicated CpGs occurring opposite methylated residues on the mother strand of the DNA, and this process is essential for the epigenetic inheritance of methylation patterns in the genome. Despite an intrinsic affinity of Dnmt1 for such hemi-methylated DNA, the molecular mechanisms that ensure the correct loading of Dnmt1 onto newly replicated DNA in vivo are not understood. The Np95 protein binds methylated CpG through its SRA domain. We have shown that localization of Np95 to replicating heterochromatin is dependent on the presence of hemi-methylated DNA. Np95 forms complexes with Dnmt1 and mediates the loading of Dnmt1 to replicating heterochromatic regions. By using Np95 deficient embryonic stem (ES) cells and embryos, we could show that Np95 is essential in vivo to maintain global and local DNA methylation and to repress transcription of retrotransposons and imprinted genes. The link between hemi-methylated DNA, Np95, and Dnmt1 thus establishes key steps of the mechanism for epigenetic inheritance of DNA methylation. Hiraoka S., Furuichi T., Nishimura G., Shibata S., Yanagishita M., Rimoin D.L., Superti-Furga A., Nikkels P.G., Ogawa M., Katsuyama K., Isono K., Toyoda H., Kinoshita-Toyoda A., Ishida N., Sanai Y., Cohn D.H., *Koseki H. *Ikegawa S. (* Corresponding authors) (2007) Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human. Nat Med. 13:1363-1367. Takada Y, Isono K, Shinga J, Turner J.M.A, Kitamura H, Ohara O, Watanabe G, Singh P.B, Kamijo T, Jenuwein T, Burgoyne P.S, Koseki H,, (2007) Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes Development 134:579-590. Fujimura Y, Isono K, Vidal M, Endoh M, Kajita H, Mizutani-Koseki Y, Takihara Y, van Lohuizen M, Otte A, Jenuwein T, Deschamps J, Koseki H (2006): Distinct roles of Polycomb group gene products between transcriptionally repressed and active domains of Hoxb8 Development 133:2371-2381 Isono K., Mizutani-Koseki Y., Komori T., Schmidt-Zachmann M.S., Koseki H (2005) Mammalian Polycomb-mediated repression of Hox genes requires the essential spliceosomal protein sf3b1. Gene Dev. 19, 536-541. 25
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