We have continued studies of chromatin structure and the regulation of eukaryotic gene expression by analysis of several ATP-dependent chromatin remodeling enzymes. This year we have further elucidated the biology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor). We completed a molecular and genetic study of Bptf, the largest subunit of NURF in the mouse, showing that Bptf is required for development of mesoderm, endoderm, ectoderm tissue lineages. We found that Bptf is required as a positive or negative regulator of several hundred genes implicated in early mouse development. This work was published in PLoS Genetics. We also participated in collaborative experiments, recently published, showing that the Drosophila NURF functions as a regulator of genes involved in innate immunity, and that an alternatively spliced form of the largest NURF subunit in Drosophila generates altered modified histone binding specificities that are critical for spermatogenesis. We have made progress on studies of the budding yeast multi-protein SWR1 complex, a member of the SWI2/SNF2 superfamily of chromatin remodeling enzymes that catalyzes the replacement of nucleosomal histone H2A with the histone H2AZ variant. We found that three out of fourteen SWR1 subunits appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit contains a second H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. This work was published in J. Biol. Chem. A collaborative project with Yawen Bais group resulted in the solution of the NMR structure of H2AZ-H2B bound to the Chz1 histone chaperone. We are developing assays to determine how the SWR1 complex is recruited to chromatin in a promoter specific manner, and to define whether one or both histone H2A molecules in a nucleosome is replaced with H2AZ. Continuing studies of the budding yeast Scm3 nonhistone protein, which binds specifically to the centromeric histone variant CenH3, and is required for assembly of CenH3 and the inner kinetochore, indicate that Scm3 binds to centromeric DNA in vitro, and may function as a structural component of centromeric chromatin. Cell cycle analyses of Scm3 and CenH3 deposition show that Scm3 binding persists throughout the cell cycle, while CenH3 is transiently lost from centromeres in the transition from G1 to S phase.
