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. 2011 Jul 19;30(16):3353–3367. doi: 10.1038/emboj.2011.241

Figure 4.

Figure 4

The kinetochore localization of the CPC and Sgo1 is impaired in H2A-I112A cells. (A–D) H2A-WT and -I112A cells containing Ipl1-3HA (A), Bir1-13Myc (B), Sgo1-3HA (C), or Bub1-3HA (D) were grown in YPAD medium containing 15 μg/ml nocodazole for 3 h at 25 °C. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA co-immunoprecipitated with the anti-Myc or anti-HA antibody (IP) were amplified with a primer set corresponding to CEN3 nucleotide sequences. The data are the average of two independent experiments. Error bars indicate the standard deviation. Dashed lines indicate the background level of the ChIP signal in an untagged strain. (E) The CPC (IPL1, SLI15, and BIR1) and SGO1 mRNA levels in nocodazole-treated H2A-WT and -I112A cells. The data are the average of two independent experiments. Error bars indicate the standard deviation. (F) The CPC (Ipl1, Sli15, and Bir1) and Sgo1 protein levels in nocodazole-treated H2A-WT and -I112A cells. (G–K) The localization of major kinetochore components was unchanged in H2A-I112A cells. The centromere-specific histone H3 variant, Cse4 (G); representative proteins of the inner kinetochore, Scm3 (H) and Mif2 (I); a representative protein of the outer kinetochore, Ctf3 (J); and a cohesin component, Scc1 (K), were analysed. Cells were subjected to ChIP as described in (A–D) with the exception that the cells were incubated in the presence of nocodazole for 1 h at 37 °C rather than at 25 °C. Since H2A-I112A cells are temperature sensitive (Supplementary Figure S6), the effect of the H2A-I112A point mutation is expected to be detectable at 37 °C by ChIP. (L) The kinetochore localization of Ipl1, a subunit of the CPC, was analysed in sgo1 cells. ChIP analysis was performed using WT and sgo1 cells containing Ipl1-3HA as described in (A–D). (M, N) The rates of missegregation (M) and mono-polar attachment (N) were increased in sgo1 cells. Under the same experimental conditions described in Figure 3B and E, the rates of missegregation and mono-polar attachment in WT and sgo1 cells were evaluated with or without prior treatment with nocodazole.