Hap2-Ino80-facilitated transcription promotes de novo establishment of CENP-A chromatin.

Puneet P Singh,Sharon A White,Manu Shukla,Christos Spanos,Juri Rappsilber,Tatsiana Auchynnikava,Pin Tong,Robin C Allshire,Alison L Pidoux,Marcel Lafos

doi:10.1101/gad.332536.119

Abstract

Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily conserved histone H3 variant, which directs kinetochore assembly and hence centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity-selected solubilized fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. Chromatin association of Hap2 is Ies4-dependent. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilizes H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.

Highlights

The accurate delivery of all chromosomes to both resulting nuclei during mitotic cell division is required for eukaryotic cell viability and to prevent aneuploidy, a hallmark of cancer (Kops et al 2005)
To identify proteins involved in the assembly of CENP-ACnp1 chromatin, GFP-tagged CENP-ACnp1 chromatin was affinity-purified from micrococcal nuclease (MNase)-solubilized chromatin extracts (Fig. 1A)
As our procedure showed association of the chaperones Scm3 (HJURP) and Sim3 (NASP) that mediate CENP-ACnp1 deposition we reasoned that other enriched, but non-centromere-specific, proteins might be involved in the incorporation of CENP-ACnp1 into centromeric chromatin

Summary

Introduction

The accurate delivery of all chromosomes to both resulting nuclei during mitotic cell division is required for eukaryotic cell viability and to prevent aneuploidy, a hallmark of cancer (Kops et al 2005). Flanking gene order is preserved, the central domain sequence is not conserved among these species Despite this lack of similarity, central domain DNA from S. octosporus and S. cryophilus, as well as S. pombe, can direct de novo CENP-ACnp and kinetochore assembly in S. pombe (Folco et al 2008; Catania et al 2015; Tong et al 2019). This suggests that these nonhomologous centromere DNAs possess innate features that program events that preferentially trigger the assembly of CENP-ACnp in place of H3 nucleosomes. It remains to be determined which transcription-associated chromatin remodeling factors provoke the replacement of H3 with CENP-A on naïve centromere DNA

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Results

Conclusion