Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing.
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 Delta asfl Delta and cac Delta hir Delta 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.
Sharp
J A
JA
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Fouts
E T
ET
Krawitz
D C
DC
Kaufman
P D
PD
D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004258:DNA Polymerase III; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D006657:Histones / Q000378:metabolism*; D018832:Molecular Chaperones; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012260:Ribonucleases; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C109731:ASF1 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C078808:HIR1 protein, S cerevisiae; C078809:HIR2 protein, S cerevisiae; D006657:Histones; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D009707:Nucleosomes; C491232:POL3 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; C533589:RLF2 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004258:DNA Polymerase III; D012260:Ribonucleases; C079009:POP2 protein, S cerevisiae