FIG. 6.
Changes to chromatin in cells lacking Vps75. (A) Steady-state histone levels are not affected in cells lacking Vps75. The relative levels of histones H2B and H3 in the wild-type (WT) or vps75Δ strain grown in YPD were determined by ChIP assays followed by quantitative PCR. Values shown are the averages with standard errors of four independent experiments and are expressed as the amounts of immunoprecipitated DNA corrected to that of input DNA. The value for the wild-type strain was set to 1 and other values expressed relative to that. (B) Deletion of VPS75 does not significantly affect the production of galactose-induced HAH2B or H3HA proteins. Wild-type (WT) or vps75Δ cells were arrested in G1 phase with α-factor for 2 h, and then galactose was added to the medium. Samples were taken at the indicated time points and cross-linked with formaldehyde. After preparation of chromatin and reversal of cross-links, proteins were fractionated on 12% polyacrylamide gels and the presence of HAH2B or H3HA proteins detected by Western blotting with an antibody directed against the HA tag (12CA5). Actin is shown as a loading control. (C) Vps75 regulates replication-independent incorporation of new histones H2B and H3. Wild-type (WT) or vps75Δ strains containing HA-tagged, galactose-inducible forms of H2B and H3, respectively, were grown in raffinose and arrested in G1 with α-factor. Cells were then treated with formaldehyde before (raffinose) or after (galactose) a 1-h induction with galactose. The relative level of HAH2B (left graph) or H3HA (right graph) was determined by ChIP assays followed by quantitative PCR. Values shown are the averages, with standard errors, of four independent experiments and are expressed as the amounts of immunoprecipitated DNA corrected to input DNA. The value for the wild-type strain in galactose was set to 1 and other values expressed relative to that. Prom, promoter; Mid, middle; TEL, telomeric DNA on chromosome 6. A one-sample t test was used to determine statistical significance, with a single asterisk denoting a P value of <0.05 and two asterisks denoting a P value of <0.01. (D) Vps75 is physically associated with the HSP30 and BSC1 genes. Wild-type (WT) yeast or a strain expressing Myc-tagged Vps75 (Tagged) were cross-linked and subjected to ChIP using an antibody against the Myc epitope. Real-time PCR was used to detect coprecipitated DNA from the promoter (Prom) and middle (Mid) of the HSP30 and BSC1 genes. Values shown are the averages, with standard errors, of three independent experiments and are expressed as amounts of immunoprecipitated DNA corrected to that of input DNA. The value for the untagged strain was set to 1 and other values expressed relative to that. (E) Histone H3 exchange rates are not significantly different at sites of Vps75 occupancy on chromatin. Peaks of Vps75 occupancy were compared to a published map of histone H3 exchange (26). A box plot comparing exchange rates at Vps75-occupied and unoccupied regions is shown. The line in the center of each box represents the median value of the distribution, and the upper and lower ends of the box are the upper (25th) and lower (75th) quartiles, respectively.