Our current projects are:

• Multiple nucleosome assembly proteins function in vivo.  Histone octamers, the fundamental repeat structures of chromatin, are assembled onto nascent DNA soon after passage of a DNA replication fork in vivo. In all eukaryotic cells, an evolutionarily conserved three-subunit protein complex termed CAF-I (Chromatin Assembly Factor-I) contributes to the first step in chromatin assembly, deposition of histones H3 and H4 onto DNA.
The CAC1, CAC2 and CAC3 genes encoding the budding yeast CAF-I subunits are not essential for viability. However, deletion of CAC genes reduces the stability of gene silencing at all heterochromatic loci: telomeres, the silent HM mating loci, and ribosomal DNA. Therefore, CAF-I plays a role in generating heterochromatin in vivo, but is not the only factor responsible for delivery of histones to DNA.
Consistent with this hypothesis, mutations in the HIR genes, which regulate histone gene transcription, cause strong synthetic reduction in heterochromatic silencing when combined with cac mutations. Another histone H3/H4 binding and deposition protein termed Asf1 is required for Hir proteins to contribute to silencing. We use recombinant proteins tools to study the mechanism of action of the Asf1/Hir assembly pathway.

• Functional dissection of yeast CAF-I.  CAF-I is recruited to sites of DNA synthesis via direct interaction with PCNA, the processivity factor for DNA polymerases. Surprisingly, mutations in cac1 that impair this interaction cause very mild silencing defects, yet combination of these alleles with asf1D or hirD gene deletions result in a dramatic loss of silencing. Consistent with these in vivo synergies, the Cac2 subunit of CAF-I physically interacts with the Asf1/H3/H4 histone deposition complex. We have generated recombinant mutant CAF-I complexes based on these results to study the mechanism of nucleosome assembly factor synergy.

• Centromeric chromatin defects in chromatin assembly mutants.  cacD hirD double mutants experience a 30-45 minute delay in cell cycle progression after DNA synthesis but prior to anaphase. The G2/M delay in cacD hirD cells is partially relieved by deletion of spindle assembly checkpoint genes, suggesting improper kinetochore function in these mutants. Consistent with this idea, cacD hirD mutants display greatly elevated rates of chromosome missegregation and genetic synergies with kinetochore mutants. Using immunolocalization and chromatin immunoprecipitation assays, we have demonstrated that a subset of CAF-I and Hir proteins in the cell are stably localized to centromeric chromatin. Furthermore, the nucleosomal structure at centromeres is perturbed in the absence of CAF-I and Hir proteins. We are currently initiating genomic approaches to determine the other, unknown sites of CAF-I proteins localization in the cell.

• CAF-I in human cells.  We are investigating the roles of CAF-I in formation of human cell chromatin by generating cell lines that express dominant negative fragments of CAF-I subunits. Transient expression of such fragments reduces the endogenous levels of CAF-I and causes dramatic cell cycle progression delays. Generation of cell lines that inducibly express these fragments is in progress.