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Background: Position-dependent gene silencing in yeast involves many factors, 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 Δ asf1 Δ and cac Δ hir Δ double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously 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 newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease–resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell 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 CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. Conclusions: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.

Background: Asf1 is a ubiquitous eukaryotic histone binding and deposition protein that mediates nucleosome formation in vitro and is required for genome stability in vivo. Studies in a variety of organisms have defined Asf1's role as a histone chaperone during DNA replication through specific interactions with histones H3/H4 and the histone deposition factor CAF-I. In addition to its role in replication, conserved interactions with proteins involved in chromatin silencing, transcription, chromatin remodeling, and DNA repair have also established Asf1 as an important component of a number of chromatin assembly and modulation complexes. Results: We demonstrate that the highly conserved N-terminal domain of S. cerevisiae Asf1 (Asf1N) is the core region that mediates all tested functions of the full-length protein. The crystal structure of this core domain, determined to 1.5 A resolution, reveals a compact immunoglobulin-like β sandwich fold topped by three helical linkers. The surface of Asf1 displays a conserved hydrophobic groove flanked on one side by an area of strong electronegative surface potential. These regions represent potential binding sites for histones and other interacting proteins. The structural model also allowed us to interpret mutagenesis studies of the human Asf1a/HIRA interaction and to functionally define the region of Asf1 responsible for Hir1-dependent telomeric silencing in budding yeast. Conclusions: The evolutionarily conserved, N-terminal 155 amino acids of histone deposition protein Asf1 are functional in vitro and in vivo. This core region of Asf1 adopts a compact immunoglobulin-fold structure with distinct surface characteristics, including a Hir protein binding region required for gene silencing.