Our group has continued studies of chromatin structure and the regulation of eukaryotic gene expression. This year we have made further progress towards understanding the biology of ATP-dependent chromatin remodeling by NURF (Nucleosome Remodeling Factor) in transgenic mice. In addition to NURF role as a regulator of TGF beta-responsive genes, NURF is also involved in the regulation of T-cell development and maturation in the thymus. We are also making significant progress in our understanding of the H2AZ incorporation into transcriptionally active chromatin. By building on earlier observations of the involvement of the SWR1 complex in that process, we were able to discover a novel nuclear chaperone Chz1 and characterized its activity and structure. Among multiple H2A-H2B chaperones, Chz1 displays specificity for H2AZ variant and we were able to demonstrate that it functions as a donor of the H2AZ-H2B dimer to SWR1 complex, which in turn catalyzes dimer's incorporation into chromatin. Analysis of in vitro assembled Chz1-H2AZ-H2B heterotrimers revealed the existence of a conserved motif which is also present in other species, thus suggesting that H2AZ-specific chaperones may be widely conserved. In eukaryotes, the segregation of chromosomes during mitosis is specified by centromere, which directs chromosome attachment, through kinetochore, to the spindle apparatus. Using a combination of biochemical, genetic and molecular cell techniques we demonstrated than yeast Scm3 nonhistone protein is necessary for the incorporation of CenH3 (centromere-specific variant of histone H3)into centromeric nuclosome and that Scm3 actually constitutes stable structural component of yeast centromere. Scm3 depletion causes loss of centromere at both structural and functional level. Moreover, reconstitution of centromerioc nucleosome core in vitro using purified recombinant protins revealed that Scm3 association with CenH3-H4 tetramer leads to the exclusion of H2A-H2B dimers. Therefore, we have proposed a new model for chromatin at yeast centromeres, involving a nonhistone protein at the core of the centromeric nucleosome.
