Factors That Promote H3 Chromatin Integrity during Transcription Prevent Promiscuous Deposition of CENP-ACnp1 in Fission Yeast

Abstract

Specialized chromatin containing CENP-A nucleosomes instead of H3 nucleosomes is found at all centromeres. However, the mechanisms that specify the locations at which CENP-A chromatin is assembled remain elusive in organisms with regional, epigenetically regulated centromeres. It is known that normal centromeric DNA is transcribed in several systems including the fission yeast, Schizosaccharomyces pombe. Here, we show that factors which preserve stable histone H3 chromatin during transcription also play a role in preventing promiscuous CENP-ACnp1 deposition in fission yeast. Mutations in the histone chaperone FACT impair the maintenance of H3 chromatin on transcribed regions and promote widespread CENP-ACnp1 incorporation at non-centromeric sites. FACT has little or no effect on CENP-ACnp1 assembly at endogenous centromeres where CENP-ACnp1 is normally assembled. In contrast, Clr6 complex II (Clr6-CII; equivalent to Rpd3S) histone deacetylase function has a more subtle impact on the stability of transcribed H3 chromatin and acts to prevent the ectopic accumulation of CENP-ACnp1 at specific loci, including subtelomeric regions, where CENP-ACnp1 is preferentially assembled. Moreover, defective Clr6-CII function allows the de novo assembly of CENP-ACnp1 chromatin on centromeric DNA, bypassing the normal requirement for heterochromatin. Thus, our analyses show that alterations in the process of chromatin assembly during transcription can destabilize H3 nucleosomes and thereby allow CENP-ACnp1 to assemble in its place. We propose that normal centromeres provide a specific chromatin context that limits reassembly of H3 chromatin during transcription and thereby promotes the establishment of CENP-ACnp1 chromatin and associated kinetochores. These findings have important implications for genetic and epigenetic processes involved in centromere specification.

The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A

“…Indeed, spt16-22 and pob3-L78R cells exhibited partial sensitivity to CENP-A Cse4 overexpression (Fig. 1C,D), similar to findings in fission yeast (Choi et al 2012). However, we were unable to detect a significant change in CENP-A Cse4 stability in these mutants (data not shown), so additional mechanisms must contribute to the underlying synthetic sickness.…”

“…Although FACT could exclude CENP-A Cse4 from euchromatin by promoting H3 incorporation (Choi et al 2012), the association of FACT with Psh1 in coimmunoprecipitation experiments suggested a more direct role in CENP-A Cse4 degradation (Krogan et al 2002;Ranjitkar et al 2010). We therefore asked whether Psh1 directly interacts with FACT in vitro.…”

“…FACT also re-establishes chromatin structure in the wake of polymerase passage, thereby maintaining epigenetic states of chromatin (Kaplan et al 2003;Jamai et al 2009). Recently, FACT and other transcription elongation factors have been shown to have an indirect role in excluding fission yeast CENP-A Cnp1 from euchromatin by reassembling H3-containing nucleosomes during transcription elongation (Choi et al 2012).…”

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin

“…To test whether FACT-dependent recycling is required for histone conservation and H4K20 methylation, we determined the levels of H4K20me1 and H4K20me3 in cells with compromised Spt16. Using an earlier data set (Choi et al 2012), we confirmed that in very highly transcribed genes (more than 10 transcripts per cell), H3 occupancy is relatively low throughout the transcribed unit (Fig. 6A).…”

“…We omitted data from very highly expressed genes because low nucleosome occupancy confounds turnover measurement (note the seemingly low turnover at the NDR in Fig. 2F; Lee et al 2004;Choi et al 2012).…”