Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci

Abstract

CAC1/RLF2 encodes the largest subunit of chromatin assembly factor I (CAF-I), a complex that assembles newly synthesized histones onto recently replicated DNA in vitro. In vivo, cac1/rlf2 mutants are defective in telomeric silencing and mislocalize Rap1p, a telomere-binding protein. Here, we report that in cells lacking CAF-I the silent mating loci are derepressed partially. MATa cac1 cells exhibit an unusual response to alpha-factor: They arrest and form mating projections (shmoos) initially, but are unable to sustain the arrest state, giving rise to clusters of shmooing cells. cac1 MATa HMLa HMRa strains do not form these shmoo clusters, indicating that derepression of HMLalpha causes the shmoo cluster phenotype in cac1 cells. When SIR3 is reintroduced into sir1 sir3 cells, HML remains derepressed indicating that SIR1 is required for the re-establishment of silencing at HML. In contrast, when SIR3 is reintroduced into cac1 sir3 cells, silencing is restored to HML, indicating that CAF-I is not required for the re-establishment of silencing. Loss of the other CAF-I subunits (Cac2p and Cac3p/Msi1p) also results in the shmoo cluster phenotype, implying that loss of CAF-I activity gives rise to this unstable repression of HML. Strains carrying certain mutations in the amino terminus of histone H4 and strains with limiting amounts of Sir2p or Sir3p also form shmoo clusters, implying that the shmoo cluster phenotype is indicative of defects in maintenance of the structural integrity of silent chromatin. MATa cac- sir1 double mutants have a synergistic mating defect, suggesting that the two silencing mechanisms, establishment and maintenance, function cooperatively. We propose a model to explain the distinctions between the establishment and the maintenance of silent chromatin.

Figure 1

CAF-I contributes to the repression of HMR. Cells with the indicated genotype at the CAC1, CAC3, RAP1, or SIR1 locus and deleted for the listed sites within HMR E::TRP1 were plated in 10-fold serial dilutions onto medium lacking tryptophan (left) or complete medium (right). Colonies were photographed after 2 days at 30°C. (Top) Strains used: WT WT, YJB959; WT orc YJB955; WT abf1, YJB1143; WT rap1, YJB1104; cac1 WT, YJB1960; cac1 orc YJB958; cac1 abf1, YJB1139; cac1 rap1, YJB1101; rap1-12 orc, YJB1638; and sir1 WT, YJB2006. (Bottom) Strains used in a separate experiment photographed after 3 days at 30°C were: cac1 orc, YJB958; cac3 orc, YJB2011; cac1 cac3 orc, YJB2009; and rap1-12 orc, YJB1638.

Figure 2

α-Factor response of cac strains on solid media. Yeast cells were spread onto α-factor–YPD plates and maintained at 23°C. Cells were analyzed at indicated times after exposure to α-factor. (A) Analysis of yeast cell populations after 18 hr on α-factor. More than 100 cells per strain were analyzed. (Shmoo) Individual cells that formed mating projections and remained arrested; (shmoo cluster) individual cells that formed multiple mating projections and eventually divided at least once; (colony) cells that formed colonies of round cells and did not appear to respond to α-factor. (Left) Strains used: WT, YJB276; cac1, YJB469; cac1 HMLa, YJB2057; sir1, YJB335; and sir1 cac1, YJB744. (Right) Strains used: WT, YJB195; cac1, YJB1838; cac2, YJB1803; cac3, YJB1581; and cac2 cac3, YJB1865. (Left) χ² tests indicated that the difference between WT and cac1 HMLa strains was not significant, whereas differences between all other pairwise combinations were significant. (Right) All pairwise combinations were significantly different except that the cac1 and the cac2 cac3 strains were not significantly different. (B) Analysis of cells over time. (Top four rows) Populations of cells; (bottom row) the same individual cells photographed at indicated times after exposure to α-factor.

Figure 3

SIR1 is required, and CAC1 is not required, for the re-establishment of HML silencing. Plasmids pSIR3 (pSE334 or pJR273) and pSIR1 (pJR910) were introduced (indicated by ⁺) into strains carrying sir3 and the other indicated mutations. Two days after transformation, transformants were allowed to mate for 18 hr with a Matα tester strain (TD1). Diploids were then selected by replica plating onto SDC medium lacking adenine and histidine. Strains used were sir3 sir1, YJB2471; sir3 cac1, YJB2109; sir3, YJB2544; WT, YJB195.

Figure 4

Mutation of CAF-I subunits causes subtle MATa mating defects. At least four individual quantitative mating assays were performed for each strain. The median value of the assays is shown. All values are normalized to the isogenic wild type. Solid bars indicate that results were statistically different from wild type at the P < 0.05 level. Strains (MATa, MATα): WT, YJB195, YJB209; cac1, YJB1838, YJB1578; cac2, YJB1803, YJB1599; cac3, YJB1581, YJB1836; cac1 cac2, YJB1804, YJB1802; cac2 cac3, YJB1865, YJB1864; cac1 cac3, YJB1862, YJB1863; sir1, YJB1940, YJB1941; sir1 cac1, YJB1962, YJB1961; sir1 cac2, YJB2000, YJB2034; sir1 cac3, YJB1945, YJB1946; sir1 cac1 cac2, YJB2044; sir1 cac2 cac3, YJB2048; sir1 cac1 cac3, YJB2007, YJB1993.

Figure 5

α-Factor response of strains with mutations in the amino termini of histones H3 or H4. Yeast cells were treated as described in Fig. 2A. χ² tests indicated that HHT HHF and HHT hhfK16R were significantly different from each other and from the other histone mutants. Strains used: HHT HHF, YJB2166; hhtΔ2-20 HHF, YJB2167; hhtΔ2-29 HHF, YJB2168; HHT hhfK5R, YJB2169; HHT hhfK5R K8R, YJB2170; HHT hhfK5R K12R, YJB2171; and HHT hhfK16R, YJB2172.

Figure 6

Limiting amounts of Sir2p or Sir3p weaken the maintenance of silencing at HML. Strains limiting for Sir2p (YJB285 [pAR14]) or Sir3p (YJB397 [pAR16]) were generated by pregrowth on raffinose, transfer to glucose for 8 hr, and then plating on α-factor lacking leucine and containing glucose. Elongated shmoo clusters that arose in these cultures are shown.

Figure 7

Model for the mechanism by which CAF-I contributes to formation of stable heterochromatin. Heterochromatin is represented as a “wall” of Sir complex proteins (Sir2p, Sir3p, and Sir4p) built on a foundation of nucleosomes (circles) composed of appropriately acetylated histones. After replication, existing nucleosomes (white circles) are randomly distributed between daughter strands of DNA. (Top) CAF-I assembles newly synthesized nucleosomes (gray circles) into chromatin. Existing Sir complex proteins (white rectangles), as well as newly synthesized Sir complex proteins (gray rectangles), associate with the nucleosomes to form a wall of proteins that restrict accessibility to the DNA. If Sir complex proteins are limiting, the wall is thinner or weaker. (Middle) If derepression of the locus occurs, nucleosomes with the “active” acetylation patterns generated during the previous cell cycle (white circles with X) form an unstable foundation that does not associate as tightly with the Sir complex proteins. In the absence of CAF-I, these nucleosomes are recycled onto daughter strands and a fragile wall of Sir complex proteins (recruited by Sir1p) is subject to “leaking” or eventual derepression. If extra Sir complex proteins are provided, the wall can become thicker and thus, more stable. (Bottom) In cac sir1 double mutants, an unstable foundation (attributable to the lack of CAF-I) and limited recruitment of Sir complex proteins (attributable to the lack of Sir1p) leads to more derepression of the locus.