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

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Singh, P.P., Shukla, M., White, S.A., Lafos, M., Tong, P., Auchynnikava, T., Spanos, C., Rappsilber, J., Pidoux, A.L., and Allshire, R.C.

Summary of Paper by Lori Koch 

In each of our cells, DNA is wrapped around many small protein complexes called nucleosomes, forming a dense structure called chromatin. One of the most important regions of each chromosome is the centromere, where a specialized nucleosome is found that ensures the chromosome is inherited properly during cell division. This specialized nucleosome contains a variant of the histone H3 protein called CENP-A. How CENP-A is enriched only at centromeres is an unsolved mystery in molecular cell biology. CENP-A protein at the centromere is epigenetically inherited, meaning it does not depend on the underlying DNA sequence but on events directed by proteins. In their recent study, researchers from Robin Allshire’s lab identified that the protein complex Ino80C regulates initial establishment of CENP-A on centromeric DNA.

First, the authors identified that subunits of the Ino80 complex were associated with CENP-A protein isolated from fission yeast cells by affinity purification and mass spectrometry. The Allshire lab researchers investigated the function of the protein Hap2, which they found associated with Ino80C and CENP-A chromatin. They found that lack of Hap2 caused lagging chromosomes during cell division, suggesting it may be involved in centromere function. Next, they found that lack of Hap2 caused a reduction in CENP-A at the centromere and the loss of an epigenetic mark around the centromere, the methylation of nucleosomes. 

To understand how CENP-A is initially localized to centromeres, the authors performed experiments with plasmid-based minichromosome DNA, which carried centromeric sequences but were not present through previous cell divisions. They found that CENP-A could not be loaded onto these “naïve centromeres” in cells lacking Hap2 as well as it was in normal cells. 

Using a dynamic protein marking strategy, the authors determined that lack of Hap2 caused reduced turnover of canonical H3 protein at both normal and ectopic centromeres. Therefore, Hap2 seems to regulate both CENP-A and canonical H3 association with DNA.   The authors hypothesized that altered transcription might affect nucleosome protein maintenance in the absence of Hap2. They measured the activity of promoters from centromere regions and found that they were not as active in the absence of Hap2. 

Overall, their study determined that Hap2-Ino80C regulates transcription from non-coding centromere DNA and the proper incorporation of CENP-A on centromeric DNA. These findings further our understanding of how centromeres are identified and inherited, a fundamental requirement for cell division.

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