Cytosine and histone methylation are important epigenetic modifications involved in the regulation of chromatin structure and gene expression. Little is known about how DNA and histone methyltransferase enzymes are controlled and targeted to appropriate genomic sites for action. This laboratory cloned CpG binding protein (CGBP), a novel factor that binds DNA containing unmethylated CpG dinucleotides, the site of cytosine methylation in mammals. This factor is also a component of the Set1 histone H3-Lys4 methyltransferase complex. Disruption of the CGBP gene leads to embryonic death in mice, and embryonic stem (ES) cells lacking CGBP are unable to differentiate in vitro and carry altered patterns of epigenetic modifications such as reduced cytosine methylation. They also contain elevated levels of histone H3-Lys4 methylation, correlated with inappropriate drifting of the Set1 protein into heterochromatin. Thus, CGBP is a regulator of both major classes of epigenetic modifications. The CGBP -/- mutant phenotype is ""rescued"" upon introduction of a CGBP-expression vector, thus offering an experimental system with which to probe structure/function relationships of CGBP. The specific aims are: (1) Determine domains of CGBP that are necessary and sufficient to rescue epigenetic modifications in CGBP -/- ES cells. Various truncated or mutated versions of CBGP will be introduced into CGBP-/- ES cells. Clones expressing physiologic levels of these CGBP variants will be examined for genomic cytosine methylation, DNA methyltransferase activity, sub-nuclear targeting of the Set1 histone methyltransferase complex, and histone H3-Lys4 and Lys9 methylation. (2) Determine domains of CGBP that are necessary and sufficient to rescue developmental potential of CGBP -/- ES cells. Transfected ES cell clones isolated in Specific Aim #1 will be assessed for developmental capacity. ES clones will be induced to differentiate in vitro by the removal of leukemia inhibitory factor from the growth media, and differentiation will be assessed by morphological examination of subsequent embryoid body formation, and by histochemical detection of alkaline phosphatase activity, a marker of stem cells. RT-PCR will also be performed to assess the expression of a panel of stem cell and lineage-restricted molecular markers. In vivo developmental potential of ""rescued"" ES cell lines will be assessed by isolating cells expressing enhanced green fluorescent protein (EGFP), injecting these cells into blastocysts, and introduction into pseudo-pregnant mice to produce chimeric animals. The contribution of ES cells to each tissue will be examined by fluorescence microscopy.
Intellectual Merit.These studies are targeted at gaining an understanding of the molecular mechanism of CGBP function. These studies will provide novel information regarding the molecular mechanisms that control the epigenetic regulation of chromatin structure. This is a fundamental question that relates to many aspects of normal development and disease.
Broader Impacts. This laboratory has a long history of integrating graduate student education with its research program. Nine doctoral students have been trained over 15 years. Ten undergraduate students, including minority students, have participated in the research as well. A similar level of student participation is anticipated for these studies.
