Fig. 2.

Yta7 regulates Cse4 levels at the centromere. (A) yta7∆ caused a reduction of centromere-bound Cse4 at CEN4 in cbf1Δ cells at both the permissive (23 °C) and restrictive (37 °C) temperatures as measured by ChIP analysis. ChIP was performed with α-HA (for Cse4) and, as a control, with an α-H4 antibody. (B) The levels of histone H3 at the centromere proper were only slightly increased in yta7∆. ChIP of myc-tagged H3 (myc-HHT2) using an α-myc antibody was used to analyze the association of H3 at CEN4 and regions located 200 bp to the left or right of CEN4. Cells were grown at 23 °C or shifted to 34 °C for 4 h prior to ChIP. Representation as in Fig. 1D. (C) yta7∆ caused reduced Cse4 levels in otherwise wild-type cells at several centromeres at 30 °C and 37 °C. Representation as in A. *P < 0.05. (D) yta7∆ caused a slight increase of H3 levels at CEN4, whereas H3 levels were strongly increased in regions surrounding the centromere. ChIP of H3 (myc-HHT2) was conducted as in B. (E) Overexpression of YTA7 caused a growth defect in the cbf1Δ strain. Serial dilution of cbf1∆ with an empty vector or a 2µYTA7 plasmid was spotted on selective medium and grown at indicated temperatures for 3 d. (F) Overexpression of YTA7 increased the levels of centromere-associated Cse4 in cbf1Δ. Representation as in A. (G) Histone H3 levels at the centromere were unaffected by YTA7 overexpression. ChIP of H3 (myc-HHT2) to the centromeric region on chromosome IV in cbf1Δ was conducted as in B. Values for ChIP in A, D, F, and G are the mean of at least three independent experiments, with error bars indicating the SD (P values by Student’s t test).