Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing

Background: Position-dependent gene silencing in yeast involves many factor s, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes), Both cac Delta asf1 Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing, However, the relationship be tween the contributions of HIR genes and ASF1 to silencing has not previous ly been explored. Results: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function, In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a new ly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micr ococcal nuclease-resistant DNA in the absence of DNA replication and stimul ated nucleosome assembly activity by recombinant yeast CAF-I during DNA syn thesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in c ell extracts, In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30 -8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents OAF-I from contributing to silencing, Conversely, other pol3 0 alleles prevented Asf1/Hir proteins from contributing to silencing. Conclusions: Yeast CAP-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote he terochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir sile ncing pathway functionally overlaps with CAP-I activity.