Histone H3 Lys79 methylation is required for efficient nucleotide excision repair in a silenced locus of Saccharomyces cerevisiae

Abstract

Methylation of specific histone lysine residues regulates gene expression and heterochromatin function, but little is known about its role in DNA repair. To examine how changes in conserved methylated residues of histone H3 affect nucleotide excision repair (NER), viable H3K4R and H3K79R mutants were generated in Saccharomyces cerevisiae. These mutants show decreased UV survival and impaired NER at the transcriptionally silent HML locus, while maintaining normal NER in the constitutively expressed RPB2 gene and transcriptionally repressed, nucleosome loaded GAL10 gene. Moreover, the HML chromatin in these mutants has reduced accessibility to Micrococcal nuclease (MNase). Importantly, chromatin immunoprecipitation analysis demonstrates there is enhanced recruitment of the Sir complex at the HML locus of these mutants, and deletion of the SIR2 or SIR3 genes restores the MNase accessibility and DNA repair efficiency at this locus. Furthermore, following UV irradiation expression of NER genes in these mutants remains at wild type levels, with the exception of RAD16 which decreases by more than 2-fold. These results indicate that impaired NER occurs in the silenced chromatin of H3K79R and H3K4,79R mutants as a result of increased binding of Sir complexes, which may reduce DNA lesion accessibility to repair enzymes.

Figure 1.

H3K79R and H3K4,79R cells are more sensitive to UV radiation than wt cells. Cells were diluted to appropriate concentrations, spread on YPD plates and irradiated at different UV doses. Colony forming ability following UV radiation was monitored and expressed as percent survival relative to unirradiated cells.

Figure 2.

NER of the HML, RPB2 and GAL10 loci in H3 methylation mutants. Cells were irradiated with 100 J/m² UV light and allowed to repair in the dark at 30°C for various times. Genomic DNA was isolated, digested with appropriate restriction enzyme(s) and subjected to T4 endonuclease V digestion. Southern analysis was performed to determine CPD removal using radioactive probes to a 2.3 kb HML fragment (A), a 3.4 kb RPB2 fragment (C) and a 2.2 kb GAL10 fragment (E). The time course of CPD removal for each strain was plotted for HML (B), RPB2 (D) and GAL10 (F) as the mean ± 1 SD for three independent experiments.

Figure 3.

Nucleosome DNA is less accessible to MNase in H3 methylation mutants. Spheroplasts were isolated from WY139 (wt) and methylation mutants, treated with different concentrations of MNase (10 U/μl stock solution) and genomic DNA was isolated, electrophoresed on agarose gels, stained with ethidium bromide, blotted and hybridized with a probe specific for the HMLα1, RPB2 and GAL10 ORF. (A) MNase digestion patterns are shown for both bulk chromatin, HML, RPB2 and GAL10 chromatin. W: WY139 and M: H3K4,79R. (B) Quantitative analysis of MNase accessibility at the three loci. Data is expressed as the ratio of mono- to tri-nucleosome signal at different concentrations of MNase. (C) Comparative scans of the 4 μl MNase lanes for the HML locus in WY139 and mutant H3K4,79R cells. (D) MNase digestion patterns for HML and RPB2 chromatin of WY139 and H3K79R. (E) Quantitative analysis of MNase accessibility at HML and RPB2. Data is expressed as the ratio of mono- to tri-nucleosome formation at different concentration of MNase.

Figure 4.

Deletion of SIR genes increases repair in the H3K4,79R mutant. The Sir2 and Sir3 genes were deleted from WY139 (wt) and H3K4,79R mutants using gene replacement, and CPD removal was measured at the HML and RPB2 loci after exposure to 100 J/m² UV light. The time courses of CPD removal are shown for the 2.3 kb HML fragment for Sir2 and Sir3 deletion mutants in (A) and (B), respectively, and the time course of CPD removal from the 3.4 kb RPB2 fragment for the Sir2 deletion mutant is shown in (C). Data represent the mean ± 1 SD for three independent experiments.

Figure 5.

MNase accessibility increases in Sir2 deletion strains. Spheroplasts were isolated from the Sir2 deletion strains of WY139 (wt) and H3K4,79R, treated with different concentrations of MNase, blotted and hybridized with a HMLα1 ORF-specific probe, as described in legend to Figure 3. (B) Quantitative analysis of MNase accessibility at the HML locus. Data is expressed as the ratio of mono- to tri-nucleosome formation at different concentration of MNase. (C) ChIP analysis, using Sir2 antibody and HMLα1 ORF primers, showing recruitment of Sir proteins at the HML locus. Chromatin immunoprecipitated from Sir2 deleted strains was used as control (right-hand panels). Each experiment was repeated three times and the data shown are for a single representative experiment. Right panel shows amplification of RPB2 gene from the ChIP DNA, which served as a control.

Figure 6.

Expression of NER genes in H3 methylation mutants after UV irradiation. Both wt and H3K4,79R cells were allowed to repair for 20 min after exposure to 100 J/m² UV radiation. Total RNA was isolated and RT-PCR was performed using gene-specific primers. Cells not treated with UV radiation served as controls. (A) Gel showing the RT-PCR products. (B) Fold change in RT-PCR product for each of the NER genes shown in (A). Data represent the mean ± 1 SD for three independent experiments.