A cassette of N-terminal amino acids of histone H2B are required for efficient cell survival, DNA repair and Swi/Snf binding in UV irradiated yeast

Abstract

The highly charged histone N-terminal domains are engaged in inter- and intra-nucleosomal interactions, and contain a host of sites used for posttranslational modification. We have studied the effect of deleting residues 30-37 from the N-terminal domain of histone H2B in yeast cells, on nucleotide excision repair (NER) following UV irradiation, as these cells are quite sensitive to UV. We find that H2B Delta30-37 cells exhibit reduced NER efficiency at three specific chromatin loci: the transcriptionally active, RPB2 locus; the transcriptionally silenced, nucleosome-loaded HML locus; and the transcriptionally repressed, non-silenced, GAL10 locus. Nuclease digestion studies indicate that H2B Delta30-37 chromatin has increased nucleosome accessibility and/or nucleosome mobility. In addition, H2B Delta30-37 mutants acquire more DNA damage, compared to wt cells, following the same dose of UV radiation. Reducing the level of damage in H2B Delta30-37 cells to match that of wt cells restores the NER rate to wt levels in the RPB2 and GAL10 loci, but NER efficiency remains low in the silenced HML locus. Interestingly, recruitment of Snf5 to the HML locus is reduced in H2B Delta30-37 cells and more transient following UV irradiation. This may reflect a lower binding affinity of the SWI/SNF complex to H2B Delta30-37 nucleosomes.

Figure 1.

NER in the HML, RPB2 and GAL10 loci. For repair experiments, cells were UV irradiated (100 J/m²) and incubated in the dark for various time periods (0.5–2 h). Genomic DNA was isolated, digested with appropriate restriction enzymes and then digested to completion with T4 endo V. Southern analyses were performed using radiolabeled probes specific for HML, RPB2 and GAL10 loci. Representative alkaline agarose gels used for CPD content (T4 endo V cutting) in the 2.3 kb Bsp1286I fragment (HML), 2.2 kb EcoRI–EcoRV fragment (GAL10) and the 3.4 kb NruI fragment (RPB2) are shown in (A, C and E), respectively. The time courses calculated for CPD removal from the HML, RPB2 and GAL10 loci are shown in (B, D and F), respectively. For each strain, data represent the mean ± 1 SD for three independent experiments.

Figure 2.

CPD formation in the HML, RPB2 and GAL10 loci. For quantification of CPDs, cells were irradiated with increasing UV doses, harvested immediately and the genomic DNA was digested to completion with T4 endo V. Southern analyses was performed using radiolabeled probes specific for RPB2, GAL10 and HML loci, respectively. Graphic-representation of the number of CPDs formed per fragment of the RPB2, GAL10 and HML loci are shown in (A, B and C), respectively. For each strain, data represent the mean ± 1 SD for three independent experiments.

Figure 3.

CPD and 6-4 photoproduct formation in total genome DNA. For total genome CPD and 6-4 photoproduct quantifications, cells were UV irradiated as mentioned in the legend to Figure 2 and the genomic DNA was subjected to slot blot hybridization with antibodies specific to CPDs or 6-4PPs. Southern analysis of the same membrane was used to correct for loading differences between slots and yield the relative number of CPDs and 6-4PPs for each strain. Graphical representation of the number of CPDs and 6-4PPs formed in the genome are shown in (A and B), respectively. For each strain, data represent the mean ± 1 SD for three independent experiments.

Figure 4.

NER in H2B Δ30–37 mutant cells with similar DNA damage levels as wt cells. H2B Δ30–37 and wt cells were irradiated at UV doses of 70 J/m² and 100 J/m², respectively, followed by repair incubations. Genomic DNA was then digested with locus specific restriction enzymes followed by T4 endo V digestions, as mentioned earlier. Graphical representation of CPD removal from the RPB2, GAL10 and HML loci are (A, B and C), respectively. For each strain, data represent the mean ± 1 SD for three independent experiments.

Figure 5.

MNase digestion patterns for H2B mutant chromatin. (A) Isolated spheroplasts from wt and H2B Δ30–37 cells were treated with different concentrations of MNase, as described in ‘Materials and Methods’ section, and genomic DNA was isolated and separated on 1.2% agarose gels, with 100 bp repeat marker fragments (New England Biolabs) run in every other lane to facilitate measurements of nucleosome repeat lengths. Digestion profiles of bulk chromatin were stained with ethidium bromide. (B) Isolated spheroplasts from wt, H2B Δ3–32, H2B Δ3–37 and H2B Δ30–37 cells were treated with different concentrations of MNase, genomic DNA was isolated and then separated on 1.2% agarose gels. M, D and T denote positions of mono-, di- and tri-nucleosome DNA populations, respectively. The position of a prominent sub-monomer band in the mutant chromatin digest is denoted with a ‘*’.

Figure 6.

Locus-specific chromatin analysis. MNase digestion patterns for (A) Bulk chromatin, stained with ethidium bromide. M, D and T denote mono-, di- and tri-nucleosome DNA populations, respectively. Southern blot hybridizations performed with probes to the (B) GAL10 (C) HML and (D) RPB2 loci, respectively.

Figure 7.

Histone occupancy at the HML locus in H2B Δ30–37 cells. ChIP was used to measure (A) histone H2B and (B) histone H3 occupancy levels at three locations within the HML locus. Error bars denote ±1 SD of the mean of three different experiments. No statistically significant differences in H2B or H3 levels were observed between wt and H2B Δ30–37 cells (P > 0.05). (C) Schematic of the yeast HML locus, including positions of nucleosomes (gray ovals) and primer sets used for ChIP experiments (horizontal lines A, B and C). Vertical arrows indicate the locations on chromosome III.

Figure 8.

Recruitment of Snf5 to specific chromatin loci during NER. ChIP analysis of Snf5 protein recruitment to distinct chromatin regions of wt and H2B Δ30–37 cells during NER. Cells were UV irradiated and incubated for different repair times. Chromatin was immunoprecipitated with antibody specific to Snf5, followed by quantitative PCR amplification using gene-specific primers. Graphical representation of Snf5 occupancy in the HML, RPB2 and PHO5 loci are shown in (A, B and C), respectively. Snf5 recruitment during repair was normalized to the Snf5 occupancy at 0 h of repair. For each strain, data represent the mean ± 1 SD for three independent experiments.

Figure 9.

Location of H2B residues 30–37 in the nucleosome core structure. A side view of the nucleosome core particle structure is shown with the positions of histone H2B residues 30–37 (colored yellow) passing in between the two gyres of the DNA superhelix (colored purple). The image was drawn using PYMOL.