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. 1998 Aug;18(8):4793-806.
doi: 10.1128/MCB.18.8.4793.

Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I

P D Kaufman  1 , J L Cohen, M A Osley
Affiliations

Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of chromatin assembly factor I

P D Kaufman et al. Mol Cell Biol. 1998 Aug.

Abstract

Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacDelta mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacDelta mutants, although hirDelta mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacDelta mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.

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Figures

FIG. 1
FIG. 1
The CAC2/HIR1 subfamily of WD repeat proteins. The yeast CAC2 gene (28), the human CAF-I p60 gene (27), the human HIRA gene (34), and the yeast HIR1 gene (34, 60) were aligned by using the PILEUP program (Genetics Computer Group, Madison, Wis.). Each protein sequence is shown starting from the initiator methionine through the seven WD motifs depicted on each line. Amino acids identical in at least three proteins are shadowed in black; conservative changes are underlined. A WD motif consensus from a large number of proteins is illustrated between repeats 3 and 4 (45), with shadowed H’s representing hydrophobic residues and DPGN representing a region that often includes those amino acids.
FIG. 2
FIG. 2
Growth defects of cacΔ hirΔ mutants. Cultures were streaked onto YPD plates for distribution into single colonies, and the plates were incubated for 3 to 5 days. (A) cacΔ hirΔ strains grown at 37°C; (B) triple and quadruple cacΔ hirΔ deletion strains grown at 30 and 37°C. Strains used were W303 (HIR+ CAC+), W303Δ1 (hir1Δ), W303Δ2 (hir2Δ), W303Δ1Δ2(FOA) (hir1Δ hir2Δ), PKY035 (cac1Δ), PKY031 (cac2Δ), PKY034 (cac3Δ), PKY102 (cac1Δ hir1Δ), PKY103 (cac2Δ hir1Δ), PKY132 (cac3Δ hir1Δ), PKY136 (cac1Δ hir2Δ ), PKY137 (cac2Δ hir2Δ), PKY103 (cac3Δ hir2Δ), PKY110 (cac1Δ hir1Δ hir2Δ), PKY111 (cac2Δ hir1Δ hir2Δ), and PKY112 (cac1Δ cac2Δ hir1Δ hir2Δ). (C) Slow growth of cac1Δ hir1Δ spores after germination. Strain PKY102 (MATα cac1Δ::LEUZ hir1Δ::HIS3) was mated to PKY090 (MATa URA3-VIIL). The resulting diploid was sporulated, and tetrads were dissected. The viable progeny, shown here after 3 days of growth on YPAD medium at 30°C, are of two sizes. Scoring of markers revealed that all of the smaller progeny were His+ Leu+, indicating that they were cac1Δ hir1Δ double mutants (data not shown). Note that the number of complete tetrads with a 3:1 ratio of large to small colonies is below the expected 2:3 frequency for tetratypes; this is presumably because of the proximity of CAC1 to the chromosome XVI centromere.
FIG. 2
FIG. 2
Growth defects of cacΔ hirΔ mutants. Cultures were streaked onto YPD plates for distribution into single colonies, and the plates were incubated for 3 to 5 days. (A) cacΔ hirΔ strains grown at 37°C; (B) triple and quadruple cacΔ hirΔ deletion strains grown at 30 and 37°C. Strains used were W303 (HIR+ CAC+), W303Δ1 (hir1Δ), W303Δ2 (hir2Δ), W303Δ1Δ2(FOA) (hir1Δ hir2Δ), PKY035 (cac1Δ), PKY031 (cac2Δ), PKY034 (cac3Δ), PKY102 (cac1Δ hir1Δ), PKY103 (cac2Δ hir1Δ), PKY132 (cac3Δ hir1Δ), PKY136 (cac1Δ hir2Δ ), PKY137 (cac2Δ hir2Δ), PKY103 (cac3Δ hir2Δ), PKY110 (cac1Δ hir1Δ hir2Δ), PKY111 (cac2Δ hir1Δ hir2Δ), and PKY112 (cac1Δ cac2Δ hir1Δ hir2Δ). (C) Slow growth of cac1Δ hir1Δ spores after germination. Strain PKY102 (MATα cac1Δ::LEUZ hir1Δ::HIS3) was mated to PKY090 (MATa URA3-VIIL). The resulting diploid was sporulated, and tetrads were dissected. The viable progeny, shown here after 3 days of growth on YPAD medium at 30°C, are of two sizes. Scoring of markers revealed that all of the smaller progeny were His+ Leu+, indicating that they were cac1Δ hir1Δ double mutants (data not shown). Note that the number of complete tetrads with a 3:1 ratio of large to small colonies is below the expected 2:3 frequency for tetratypes; this is presumably because of the proximity of CAC1 to the chromosome XVI centromere.
FIG. 2
FIG. 2
Growth defects of cacΔ hirΔ mutants. Cultures were streaked onto YPD plates for distribution into single colonies, and the plates were incubated for 3 to 5 days. (A) cacΔ hirΔ strains grown at 37°C; (B) triple and quadruple cacΔ hirΔ deletion strains grown at 30 and 37°C. Strains used were W303 (HIR+ CAC+), W303Δ1 (hir1Δ), W303Δ2 (hir2Δ), W303Δ1Δ2(FOA) (hir1Δ hir2Δ), PKY035 (cac1Δ), PKY031 (cac2Δ), PKY034 (cac3Δ), PKY102 (cac1Δ hir1Δ), PKY103 (cac2Δ hir1Δ), PKY132 (cac3Δ hir1Δ), PKY136 (cac1Δ hir2Δ ), PKY137 (cac2Δ hir2Δ), PKY103 (cac3Δ hir2Δ), PKY110 (cac1Δ hir1Δ hir2Δ), PKY111 (cac2Δ hir1Δ hir2Δ), and PKY112 (cac1Δ cac2Δ hir1Δ hir2Δ). (C) Slow growth of cac1Δ hir1Δ spores after germination. Strain PKY102 (MATα cac1Δ::LEUZ hir1Δ::HIS3) was mated to PKY090 (MATa URA3-VIIL). The resulting diploid was sporulated, and tetrads were dissected. The viable progeny, shown here after 3 days of growth on YPAD medium at 30°C, are of two sizes. Scoring of markers revealed that all of the smaller progeny were His+ Leu+, indicating that they were cac1Δ hir1Δ double mutants (data not shown). Note that the number of complete tetrads with a 3:1 ratio of large to small colonies is below the expected 2:3 frequency for tetratypes; this is presumably because of the proximity of CAC1 to the chromosome XVI centromere.
FIG. 3
FIG. 3
Deletion of both CAC and HIR genes synergistically reduces silencing of a telomere-proximal URA3 gene, the ADE2 gene at HMR, and the a genes at the natural HML locus. (A) Quantitation of silencing in representative strains. PKY090 (CAC+), PKY106 (cac1Δ), PKY117 (hir1Δ), PKY361 (spt21Δ), PKY329 (spt21Δ hir1Δ), PKY360 (cac1Δ spt21Δ), PKY302 (cac1Δ hir1Δ), and JRY4470 (sir2Δ) were used. Tenfold serial dilutions of each strain were spotted onto nonselective (YPAD) medium to observe the total number of cells and onto FOA medium to observe cells capable of telomeric silencing. For each independent experiment, the fraction of viable cells resistant to FOA (FOAR) was normalized to the value obtained for the wild-type (wt) strain; the averages of data from three experiments ± standard deviations are shown. <, no FOA-resistant colonies were observed (of 2 × 106 to 4 × 106 plated). (B) Strains containing the HMR+::ADE2 allele (68) were grown on YPD medium without additional adenine. A dark colony color indicates silencing of the ADE2 gene. Strains PKY268 (wild type [wt]), PKY269 (cac1Δ), and PKY375 (cac1Δ hir1Δ) were used. (C) Strains PKY730 (MATa hm1Δ sir1Δ cac2Δ hir3Δ) (left) and PKY364 (MATa HMLα sir1Δ cac2Δ hir3Δ) (right) were replica plated onto YPD medium to test for growth (upper panel) or onto synthetic dextrose medium spread with a lawn of mating tester strain 217 (MATα his1) to select for diploid formation (lower panel).
FIG. 4
FIG. 4
Increased accessibility of dam methylase to telomeric chromatin in a cac2Δ hir1Δ double mutant. (A) Diagram of the URA3 gene adjacent to the chromosome VIIL telomere [(TG)n] and the ura3-1 gene at the natural locus on chromosome V (33, 78). Restriction sites for HindIII and 5′-GATC-3′ sequences are indicated by H3 and Sau, respectively. The GATC sequence that is more protected from methylation in wild-type cells than in sir cells is indicated by Sau∗. Expected restriction fragments (A, B, C, and the telomeric fragment T) are shown schematically. (B) Strains expressing bacterial dam methylase were PKY300 (CAC+), PKY299 (cac1Δ), PKY305 (cac2Δ), PKY310 (cac2Δ hir1Δ), PKY311 (hir1Δ), and AJL387-5aΔsir2 (sir2Δ). DNA from each strain was digested with four different combinations of restriction enzymes prior to agarose gel electrophoresis and DNA hybridization with a URA3 probe. Shown from left to right, for each strain: digestion with HindIII alone, digestion with HindIII plus DpnI, digestion with HindIII plus DpnII, digestion with HindIII plus Sau3AI. “DpnI protection” indicates the fraction of total counts in the HindIII plus DpnI lanes present in the B restriction fragment, indicating protection of the GATC site internal to the telomeric URA3 gene from dam methylase. The values presented are averages of data from two independent experiments.
FIG. 5
FIG. 5
Changes in histone gene dosage and expression affect telomeric gene silencing in a cacΔ mutant but not in wild-type cells. Telomeric silencing of the URA3-VIIL reporter was assayed as described in the legend to Fig. 3. For each strain, the fraction of viable cells resistant to FOA (FOAR) was normalized to the value obtained for the wild-type strain; the averages of values from n experiments ± the standard deviations are shown, except for the hhf2Δ cac2Δ strain, for which the averages of data from two experiments are presented. (A) Deletion of histone H3 and H4 genes. Strains PKY090 (wild type [wt]), PKY107 (cac2Δ), PKY408 [(hht1-hhf1)Δ], PKY409 [(hht1-hhf1)Δ cac2Δ], PKY410 [(hht2-hhf2)Δ], PKY411 [(hht2-hhf2)Δ cac2Δ), PKY412 (hhf2Δ), and PKY413 (hhf2Δ cac2Δ) were used. (B) Deletion of the HTA2-HTB2 gene pair encoding histones H2A and H2B. Strains PKY090 (wt), PKY106 (cac1Δ), PKY499 [(hta2-htb2)Δ], and PKY500 (hta2-htb2)Δ cac1Δ] were used. (C) Deletion of the negative regulatory site in the HTA1-HTB1 promoter. Strains PKY090 (wt), PKY106 (cac1Δ), PKY581 (HTA1Δneg), and PKY582 (HTA1Δneg cac1Δ) were used.
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References

    1. Aparicio O M, Billington B L, Gottschling D E. Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae. Cell. 1991;66:1279–1287. - PubMed
    1. Chang L, Loranger S S, Mizzen C, Ernst S G, Allis C D, Annunziato A T. Histones in transit: cytosolic histone complexes and diacetylation of H4 during nucleosome assembly in human cells. Biochemistry. 1997;36:469–480. - PubMed
    1. Clark-Adams C D, Norris D, Osley M A, Fassler J S, Winston F. Changes in histone gene dosage alter transcription in yeast. Genes Dev. 1988;2:150–159. - PubMed
    1. Compagnone-Post, P. A., J. Recht, and M. A. Osley. Unpublished data.
    1. Dilworth S M, Black S J, Laskey R A. Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts. Cell. 1987;51:1009–1018. - PubMed

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