Analysis of a mutant histone H3 that perturbs the association of Swi/Snf with chromatin - PubMed (original) (raw)

Analysis of a mutant histone H3 that perturbs the association of Swi/Snf with chromatin

Andrea A Duina et al. Mol Cell Biol. 2004 Jan.

Abstract

We have isolated new histone H3 mutants in Saccharomyces cerevisiae that confer phenotypes indicative of transcriptional defects. Here we describe the characterization of one such mutant, encoded by the hht2-11 allele, which contains the single amino acid change L61W in the globular domain of H3. Whole-genome expression analyses show that the hht2-11 mutation confers pleiotropic transcriptional defects and that many of the genes it affects are normally controlled by the Swi/Snf chromatin remodeling complex. Furthermore, we show that Swi/Snf occupancy at two promoters, PHO84 and SER3, is reduced in hht2-11 mutants. Detailed studies of the PHO84 promoter suggest that the hht2-11 mutation impairs Swi/Snf association with chromatin in a direct fashion. Taken together, our results strongly suggest that the integrity of the globular domain of histone H3 is an important determinant in the ability of Swi/Snf to associate with chromatin.

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Figures

FIG. 1.

Location and nature of the amino acid substitution encoded by the hht2-11 allele. (A) Side view of the yeast nucleosome core particle as solved by White et al. (62). Part of the DNA is removed for clarity. The histone proteins are depicted as follows: H2A is in gold, H2B is in red, H3 is in blue, and H4 is in green. The arrow indicates the location of 61L within one of the two histone H3 proteins. (B and C) Close-up views of the region of interest showing the wild-type structure (B) and the predicted structure modeled with the L61W substitution (C). These figures were kindly provided by Cindy L. White and Karolin Luger.

FIG. 2.

Phenotypic analyses of strains harboring the hht2-11 allele. (A) Phenotypes conferred by hht2-11 when present as the sole source of histone H3. The mutant strains used were as follows: HHT2 (strain FY2163), hht2-11 (FY2164), 2μm hht2-11 (FY2165), _snf2_Δ (FY2170), and _snf2_Δ hht2-11 (FY2234). Cells were grown to saturation overnight in YPD medium, washed with H2O, and then spotted in a dilution series from 7 × 106 to 7 × 103 cells/ml on the indicated medium (see Materials and Methods). For some phenotypes, only the two most concentrated spots are shown for simplicity. An Spt− phenotype corresponds to growth on SC−Lys medium (see Table 2). The plates were then incubated at either 30°C or at the indicated temperature for the following times: YPD, 2 days; YPD at 37°C, 2 days; YPD at 14°C, 11 days; YPD + HU, 7 days; YPD + caffeine (Caff.), 5 days; YPD + formamide (Form.), 3 days; SD, 3 days; SD−inositol (ino), 4 days; and SC−Lys, 3 days. (B) Dominant phenotypes conferred by hht2-11. The strains used are as follows: HHT1 HHT2 (strain FY84), HHT1 hht2-11 (strain FY2160), and _HHT1 hht2_Δ (strain FY2161). Cells were grown and spotted as described above, except that for “SC-Lys” and “YPD 14°C Caff.” the spots shown are at concentrations of 8 × 107 cells/ml. The plates were then incubated as described above, except for the “YPD 14°C Caff. plate,” which was incubated for 30 days.

FIG. 3.

Overlap in the number of genes whose expression is affected by the hht2-11 and _snf2_Δ mutations. (A) A Venn diagram showing the number of genes whose expression is either increased or decreased in hht2-11 and _snf2_Δ cells grown at 30°C and the overlap in the two data sets with the corresponding P value. The data from 3,639 genes were used for this comparison. (B) Same as for panel A, except that the data for the hht2-11 mutant was obtained from experiments in which the cells were shifted to 14°C. The data from 2,955 genes were used for this comparison.

FIG. 4.

The hht2-11 and _snf2_Δ mutations affect SER3 and PHO84 expression in a similar manner. (A) Northern analysis of SER3 mRNA levels in HHT2 (strain FY2163), hht2-11 (strain FY2164), _snf2_Δ (strain FY2169), and _snf2_Δ hht2-11 (strain FY2171) strains. _snf2_Δ and _snf2_Δ hht2-11 cells were grown at 30°C, whereas the HHT2 and hht2-11 cells were either grown at 30°C or shifted to 14°C for 24 h, as indicated. The levels of the SNR190 transcript, used as a loading control, are also shown for each strain. The data shown is representative of at least three independent experiments. (B) Northern analysis of PHO84 mRNA levels was performed with the same strains and conditions as described for panel A. The data shown are representative of at least three independent experiments.

FIG. 5.

Association of the Swi/Snf complex with the SER3 and PHO84 promoters is perturbed by the hht2-11 mutation. (A) Chromatin immunoprecipitations were performed on HHT2 (strain FY2166) and hht2-11 (strain FY2167) strains expressing the Snf5-Myc protein. A strain lacking the Snf5-Myc fusion (no tag, strain FY2168) was used as negative control. Amplifications of the SER3 promoter and the POL1 region (used as an internal control) were performed on chromatin samples prior to immunoprecipitation (IN, input) and after immunoprecipitation with the A14 anti-Myc antibody (IP, immunoprecipitate). For each sample, two dilutions (2× and 1×) of the chromatin templates were used for the PCR amplification to ensure linearity. The percent immunoprecipitation values (%IPs) of SER3 and POL1 were measured, and the ratios are shown in the bar graph. Each value represents the average ratio of the SER3 %IP to the POL1 %IP with the standard error from three independent experiments. (B) Chromatin immunoprecipitations were performed as described in panel A, except that the PCR amplifications were directed toward the promoter of the PHO84 gene instead of SER3. The bar graph shows the average ratios of the PHO84 %IP to the POL1 %IP with the standard error for each strain measured in three independent experiments.

FIG. 6.

H3 L61W causes chromatin changes at the SER3 promoter. Chromatin was isolated from HHT2 (strain FY2201), hht2-11 (strain FY2202), and _snf2_Δ (strain FY2203) strains and treated with increasing amounts of MNase, as indicated. The resulting material was subjected to indirect end-labeling analysis. All strains were deleted for the SER33 gene to prevent cross-hybridization with the SER3 probe. Part of the SER3 gene and upstream region is depicted on the left. The brackets indicate the chromatin regions most clearly affected by both the hht2-11 and the _snf2_Δ mutations. Sites that become more sensitive to MNase digestion in either mutant are indicated with closed circles, whereas sites that become more resistant are indicated with open circles. N, naked DNA controls.

FIG. 7.

Chromatin association of Pho4 at PHO84 is decreased in hht2-11 and _snf2_Δ cells, but the effects of the two mutations are not additive. Chromatin immunoprecipitations were performed and are presented as described in Fig. 5B, except that the chromatin samples were immunoprecipitated by using a polyclonal antibody directed against the Pho4 protein. The strains used were as follows: HHT2 (strain FY2166), hht2-11 (strain FY2167), _snf2_Δ HHT2 (FY2169), _snf2_Δ hht2-11 (strain FY2171), and _pho4_Δ (strain FY2235). To ensure that the enrichment of the PHO84 signal detected is specific for the presence of Pho4 at the promoter, we performed control immunoprecipitations with either Pho4 antibody added to _pho4_Δ cells (first set of experiments shown here) or no Pho4 antibody added to PHO4 cells (data not shown). In both cases, no enrichment for the PHO84 promoter was observed compared to the POL1 internal control. The %IP values for the PHO84 and POL1 regions were measured, and the average values and corresponding standard errors from at least three independent samples for each genotype are shown in the bar graph.

FIG. 8.

The hht2-11 mutation affects expression of SER3 and PHO84 to a greater degree than does an _arg82_Δ mutation. (A) Northern analysis of SER3 levels in HHT2 (strain FY2163), hht2-11 (FY2164), and _HHT2 arg82_Δ (FY2172) cells. The levels of SNR190 mRNA were measured and used as a normalization control. Shown here is one representative result from three independent experiments. (B) Northern analysis of PHO84 levels was performed as described in panel A.

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Analysis of a mutant histone H3 that perturbs the association of Swi/Snf with chromatin - PubMed (original) (raw)