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. 2009 Jan 9;284(2):740-50.
doi: 10.1074/jbc.M806274200. Epub 2008 Nov 18.

Saccharomyces cerevisiae linker histone Hho1p functionally interacts with core histone H4 and negatively regulates the establishment of transcriptionally silent chromatin

Qun Yu  1 , Holly Kuzmiak, Yanfei Zou, Lars Olsen, Pierre-Antoine Defossez, Xin Bi
Affiliations

Saccharomyces cerevisiae linker histone Hho1p functionally interacts with core histone H4 and negatively regulates the establishment of transcriptionally silent chromatin

Qun Yu et al. J Biol Chem. .

Abstract

Saccharomyces cerevisiae linker histone Hho1p is not essential for cell viability, and very little is known about its function in vivo. We show that deletion of HHO1 (hho1Delta) suppresses the defect in transcriptional silencing caused by a mutation in the globular domain of histone H4. hho1Delta also suppresses the reduction in HML silencing by the deletion of SIR1 that is involved in the establishment of silent chromatin at HML. We further show that hho1Delta suppresses changes in silent chromatin structure caused by the histone H4 mutation and sir1Delta. These results suggest that HHO1 plays a negative role in transcriptionally silent chromatin. We also provide evidence that Hho1p hinders the de novo establishment of silent chromatin but does not affect the stability of preexistent silent chromatin. Unlike canonical linker histones in higher eukaryotes that have a single conserved globular domain, Hho1p possesses two globular domains. We show that the carboxyl-terminal globular domain of Hho1p is dispensable for its function, suggesting that the mode of Hho1p action is similar to that of canonical linker histones.

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Figures

FIGURE 1.
FIGURE 1.
HHO1 functionally interacts with histone H4 and negatively regulates transcriptional silencing. A, phenotypes of the Y88G mutant and their suppression by hho1Δ. Shown are growth phenotypes of strains 1–8 under different conditions. Odd numbered strains are wild type (WT) for HHO1 (+), whereas even numbered ones are hho1Δ (–). Cells of each strain were grown to late log phase, and serial 10-fold dilutions were spotted on four synthetic complete (SC) plates and allowed to grow for 3 days under different conditions as indicated. FOA, SC supplemented with 1 mg/ml 5-fluoroorotic acid. MMS, SC with 0.02% methyl methanesulfonate. The histone H4 (HHF1) alleles are indicated on the left, and strain numbers are on the right. The silencing reporter construct is illustrated above the FOA panel. B, levels of the transcripts of genes SIR1 through SIR4 in H4-Y88G and hho1Δ single and double mutants as measured by RT-PCR. RNAs were isolated from strains 1–4 grown to log phase and used as template for RT-PCR with five pairs of primers for SIR1 through SIR4 and ACT1, respectively. The PCR products were analyzed by agarose gel electrophoresis. C, HHO1 deletion suppresses the effect of H4-Y88G on HMR silencing. Shown are growth phenotypes of strains 9–12 on SC and FOA media. Each strain bears a modified HMR locus with the URA3 reporter gene inserted to the left of an inverted HMR-E silencer, as shown at the top. D, HHO1 deletion suppresses the effect of H4-Y88G on HML silencing. Shown are growth phenotypes of strains 13–16 on SC and FOA media. The silencing reporter construct is illustrated at the top. E and F, HHO1 is a high copy inhibitor of HM silencing. Shown are growth phenotypes of strains 17–20 on –Leu (SC minus leucine) and –Leu+FOA media.
FIGURE 2.
FIGURE 2.
HHO1 suppresses the alteration in silent chromatin structure induced by the H4-Y88G mutation. A, effects of H4-Y88G and hho1Δ single and double mutations on the supercoiling of HML DNA. The modified HML locus in strains 21–25 is illustrated at the top. Two FRTs bracket the HML locus, excluding the HML-E and -I silencers. Cells grown in YPR medium (1% yeast extract, 2% bacto-peptone, and 2% raffinose) were treated with galactose (2%) for 2.5 h to induce the expression of PGAL10-FLP1. Nucleic acids were isolated and fractionated on agarose gels supplemented with 13 μg/ml chloroquine. Under this condition, more negatively supercoiled circles migrate more slowly (39). After Southern blotting, the topoisomers of the HML circle were detected by an HML-specific probe. The nicked form of the HML circle is indicated by N. The profiles of topoisomers in lanes 1–5 were determined by using the NIH Image software and are shown at the bottom. The center of distribution of topoisomers in each lane is marked by an arrowhead. B, effects of H4-Y88G and hho1Δ single and double mutations on the supercoiling of the 2 μm plasmid. DNA from strains 1–4 grown in YPD (1% yeast extract, 2% bacto-peptone, and 2% glucose) was analyzed by agarose gel electrophoresis in the presence of chloroquine. The nicked and linear forms of the plasmid are indicated by N and L, respectively.
FIGURE 3.
FIGURE 3.
Association of Hho1p with silent and active chromatin regions. A, schematics of the HMR and HML loci and flanking sequences as well as the subtelomeric region of the right arm of chromosome VI. The HMR-E and -I silencers, HML-E and -I silencers, and the W, X, Ya, Yα, and Z elements at the HM loci are indicated. The locations of DNA segments a to f tested in ChIP are indicated as black bars. Tandem arrowheads, telomeric repeats. B, ChIP analysis of the abundance of Hho1p-Myc around HMR, HML, and Tel VI-R. The abundance of sequences a–f as well as a control sequence at the ACT1 locus in the immunoprecipitated chromatin fragments was measured by PCR before (Input) and after (α-Myc) chromatin IP. No Ab, samples from mock IP without antibody. Three independent ChIP experiments (designated 1–3 for the H4 strain 26, and 1′–3′ for the H4-Y88G strain 27) were performed. C, quantification of the ChIP data shown in A. See “Results” for a description.
FIGURE 4.
FIGURE 4.
HHO1 deletion suppresses the silencing defect caused by sir1Δ. A, shown are growth phenotypes of MATa strains 28–31 on SC medium (Growth) and synthetic minimum medium lacking amino acids coated with cells of the MATα tester strain DC17 (MATα his1)(Mating). Only diploid cells resulting from the mating of the MATa and MATα cells can growth on the Mating plate. The relevant genotype of each strain is indicated on the left, and strain number is on the right. B, effects of sir1Δ and hho1Δ single and double mutations on the supercoiling of HML DNA. The modified HML locus in strains 32–36 is illustrated at the top. Two FRTs bracket the HML locus, including the HML-E and -I silencers. DNA samples isolated after the induction of the excision of the HML circle were analyzed by agarose gel electrophoresis in the presence of 13 μg/ml chloroquine. The nicked form of the HML circle is indicated by N. The topoisomers of HML from wild type and sir3Δ strains are designated SIR+ and sir–, respectively.
FIGURE 5.
FIGURE 5.
HHO1 negatively regulates the de novo establishment of transcriptionally silent chromatin. A, strategy for examining the de novo establishment of silent chromatin on the HML circle. Shaded and filled circles denote nucleosomes in active chromatin and silent chromatin, respectively. See“Results” for a description. B, examination of the kinetics of establishment of silent chromatin on the HML circle in strains 37 (sir3-8 HHO1) and 38 (sir3-8 hho1Δ). Cells of each strain grown at 30 °C in YPR medium were shifted to galactose medium and incubated for 2.5 h to induce excision of the HML circle. Cells were then shifted to YPD medium and incubated at 23 °C for up to 20 h. Aliquots of the culture were harvested at time points 0, 1, 2.5, 4.5, 7.5, and 20 h. DNA was isolated from all samples and fractionated by agarose gel electrophoresis in the presence of 13 μg/ml chloroquine. N and L, nicked and linear forms of the HML circle, respectively. The topoisomers in the SIR3 wild type strain 32 and sir3Δ strain 36 are designated SIR+ and sir–, respectively. C, the profiles of topoisomers in lanes 1, 4–6, 7, and 10–14 were determined by NIH Image. The center of distribution of topoisomers in lanes 13 (SIR+) and 14 (sir–) as well as the position of the linear form of the circle (L) are marked by shaded lines.
FIGURE 6.
FIGURE 6.
HHO1 does not affect the stability of preexistent silent chromatin. A, strategy for examining the stability of silent chromatin on silencer-free HML circles. Shaded and filled circles denote nucleosomes in active chromatin and silent chromatin, respectively. See “Results” for a description. B, examination of the kinetics of the conversion of HML silent chromatin to active chromatin after its excision from the genome in strains 21 (HHO1) and 23 (hho1Δ). Cells of each strain grown to log phase in YPR were treated with galactose for 2.5 h to induce excision of the HML circle. Cells were then shifted and diluted into YPD medium and incubated for 9 h. Aliquots of the culture were harvested at time points 0, 2, 3, 5, and 9 h. DNA was isolated from the samples and fractionated by agarose gel electrophoresis in the presence of chloroquine. N and L, nicked and linear forms of the HML circle, respectively. Topoisomers of the HML circle in the sir3Δ strain 31 are designated sir–. The asterisk indicates cross-hybridizing DNA.
FIGURE 7.
FIGURE 7.
Hho1p functionally resembles tripartite linker histones in metazoans. A, schematics of the structures of tripartite linker histone H1 and yeast Hho1p. N, G, and C, the NH2-terminal, globular, and COOH-terminal domains, respectively. C′, the sequence between GI and GII domains of Hho1p. B, GII of Hho1p is dispensable for Hho1p function. Shown are growth phenotypes of strains 39–43 under different conditions. The schematic of the HHO1 allele in each strain is shown on the left. C, Western blotting analysis of full-length and truncated Hho1p proteins. Protein extracts from strains 45–49 were run on SDS-PAGE and strained with Coomassie Blue (right) or probed with an anti-Hho1p antiserum (left). The antiserum was raised against both a peptide from the N domain of Hho1p and a peptide from GII and is therefore able to detect both NH2- and COOH-terminally truncated Hho1p proteins (28). D, Xenopus linker histone H1° and human H1.1 ectopically expressed in yeast have effects on silencing similar to those of Hho1p. Shown are growth phenotypes of strains 39, 40, 44, and 45 on –Trp and –Trp+FOA media. The silencing reporter construct in these strains is illustrated at the top.
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