GSE98396
Analysis of gene expression by RNA Seq in mutants of Psh1, Cac2, or the double mutant relative to WT upon overexpression of Cse4 in Saccharomyces cerevisiae.
Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could lead to ectopic centromere formation and missegregation, and could affect DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into noncentromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. Yeast CAF-1 (yCAF-1) is a heterotrimeric protein complex consisting of CAC1, CAC2, and CAC3, which interacts with Cse4, and can assemble Cse4 nucleosomes in vitro. yCAF-1 regulates the stability of both soluble and chromatin associated Cse4. Loss of yCAF-1 can rescue growth defects and changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin. Furthermore, the misincorporation of CenH3 at promoter regions may have negative consequences for gene expression. Overall design: Examination of gene expression by RNA Seq in yeast mutants of Psh1, Cac2, or the double mutant relative WT in duplicate using Illumina sequencing