GSE129195
Genomewide Localization of Yeast CENP-A (Cse4) in Wild-type, met30-6, and cdc4-1 Strains
The evolutionarily conserved F-box proteins Met30 and Cdc4 interact with Cse4 in vivo and cooperatively regulate cellular levels of Cse4 by ubiquitin-mediated proteolysis. To survey genomewide localization of Cse4 in met30-6 and cdc4-1 mutants, chromatin immunoprecipitation (ChIP) was carried out in strains expressing HA-tagged Cse4 from its endogenous promoter. Prominent Cse4 peaks were observed only at centromeres (CENs) in all three strains, and a statistically significant increase of Cse4 enrichment at CENs was observed in the mutants. Though extracentromeric peaks of Cse4 accumulation are detected in both wild-type and mutant strains, peak enrichment (vs. the 10-kb local background) in these regions is much lower than that observed for any of the 16 centromeres, and there is no statistically significant difference in Cse4 enrichment at non-CEN peaks between wild-type and mutants. This analysis reveals that the increased mislocalization of Cse4 in met30-6 and cdc4-1 strains detected by Cse4 staining on chromosome spreads is not the result of high levels of enrichment at discrete extracentromeric loci; rather, endogenously expressed Cse4 in met30-6 and cdc4-1 strains accumulates at low levels across the genome. Overall design: Cse4 ChIP-seq was carried out in wild-type, met30-6, and cdc4-1 yeast strains. Replicate samples of input and immunoprecipitated DNA were analyzed. Untagged control strains were included as additional controls.