Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast

Abstract

Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, 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 non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some 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.

Reduced Gene Dosage of Histone H4 Prevents CENP-A Mislocalization inSaccharomyces cerevisiae

“…However, the polyploid nature of the arp8Δ strain precluded further study with this suppressor. The stringent growth conditions of the screen also prevented the identification of deletion of Cac2, a subunit of the CAF-1 complex, which promotes Cse4 incorporation at noncentromeric regions (HEWAWASAM et al 2018). We determined that cac2Δ cannot suppress the psh1Δ GALCSE4 SDL under the conditions used in our screen (data not shown).…”

“…In these strains, expression of Scm3 is regulated by a galactose-inducible promoter and is only expressed when grown in galactose medium, but not in glucose medium. However, overexpression of Cse4 from a copper-inducible promoter can suppress the growth defect caused by depletion of Scm3 on copper-containing medium (HEWAWASAM et al 2018). We constructed hhf2Δ GAL-SCM3 Cu-CSE4 strains and performed Western blot analysis to confirm the induced overexpression of Cse4 in these strains when grown in copper-containing medium ( Figure 3D).…”

“…The evolutionarily conserved CENP-A-specific histone chaperones (Scm3 in S. cerevisiae and S. pombe, CAL1 in D. melanogaster, Holliday Junction Recognition Protein HJURP in humans) mediate the centromeric localization of CENP-A (CAMAHORT et al 2007;MIZUGUCHI et al 2007;STOLER et al 2007;FOLTZ et al 2009;PIDOUX et al 2009;WILLIAMS et al 2009;SHUAIB et al 2010;CHEN et al 2014). In budding yeast, other chaperones such as Chromatin Assembly Factor 1 (CAF-1), an evolutionarily conserved replication-coupled histone H3/H4 chaperone, can facilitate the deposition of overexpressed Cse4 when Scm3 is depleted (HEWAWASAM et al 2018). The CAF-1 orthologues Mis16 in S. pombe and RbAp46/48 in humans and D. melanogaster also contribute to centromeric localization of CENP-A (FUJITA et al 2007;PIDOUX et al 2009;WILLIAMS et al 2009;BOLTENGAGEN et al 2016).…”

“…Studies from our laboratory and those of others show that the transcription-coupled histone H3/H4 chaperone DAXX/ATRX promotes mislocalization of CENP-A to non-centromeric regions in human cells (LACOSTE et al 2014;SHRESTHA et al 2017). In budding yeast, CAF-1 contributes to the mislocalization of overexpressed Cse4 to non-centromeric regions (HEWAWASAM et al 2018).…”

“…We have recently shown that sumoylation of Cse4K215/216 in the C-terminus of Cse4 facilitates its interaction with CAF-1 and this promotes the deposition of Cse4 to non-centromeric regions (OHKUNI et al 2020). Notably, psh1∆ cac2∆ GALCSE4 strains and psh1∆ GALcse4K215/216R/A strains do not exhibit SDL due to reduced mislocalization of Cse4 (HEWAWASAM et al 2018;OHKUNI et al 2020).…”