FIG. 2.

Vps75 is physically associated with sites of active transcription, and its expression remains constant throughout the cell cycle. (A) Vps75 is recruited to GAL10 following galactose induction. Cells expressing a tagged form of Vps75 were grown in medium containing raffinose at 30°C, and GAL10 was induced by the addition of galactose for 1 h. Glucose was then added and the cells grown for a further 20 min to repress GAL10. The occupancy of Vps75 at the promoter (prom), 5′ end (5′), middle (mid), and 3′ end (3′) of GAL10 in raffinose, galactose, or glucose was measured by ChIP followed by quantitative PCR. Occupancy in raffinose was set to 1. Values shown are the averages, with standard errors, of four independent experiments. (B) Vps75 is recruited to HSP104 following heat shock. Wild-type (WT) or rtt109Δ cells expressing a tagged form of Vps75 were grown in YPD at 30°C, switched to 39°C for 10 min to induce HSP104, and then returned to room temperature for 15 min [+15 (RT)] to repress the gene. At the indicated time points, cells were cross-linked and the level of Vps75 at the HSP104 promoter (Prom) or ORF (Mid) was measured by ChIP followed by quantitative PCR. Occupancy prior to heat shock (0 min) was set to 1. Values shown are the averages, with standard errors, of three independent experiments. (C) Vps75 is recruited to PHO5 following induction by phosphate starvation. Wild-type (WT) or rtt109Δ cells expressing a tagged form of Vps75 were grown in YPD, spun down, and resuspended in either YPD (+Pho) or medium lacking phosphate (−Pho). After 2 h of induction, the occupancy of Vps75 at the promoter (Prom), 5′ end (5′), middle (Mid), and 3′ end (3′) of PHO5 was measured as described above. Occupancy in YPD was set to 1. Values shown are the averages, with standard errors, of four independent experiments. (D) Expression of Vps75 does not change during the cell cycle. A strain expressing Vps75-Myc was arrested in G₁ with α-factor and then released. Samples were taken at the indicated time points for Western analysis. Clb2 is shown as a cell cycle-regulated G₂-M marker, and actin as a loading control. (E) Vps75 can be cross-linked to Rpb1. Cells from a Vps75-Myc (T) or untagged wild-type (WT) strain were cross-linked with formaldehyde and chromatin prepared as for ChIPs. Extracts were immunoprecipitated with 9E11 (anti-Myc) and coprecipitating Rpb1 identified by Western analysis following reversal of cross-links.