Abstract
Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling.
Highlights
Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases
New dCENP-A can be targeted to sites of previous CENP-A deposition by its chaperone CAL1, which is recruited to centromeres by dCENP-C60,61
Loading of new CENP-A is restricted to mitosis and G15–8 and serves primarily to replenish CENP-A containing nucleosomes that became diluted by half during the preceding S-phase
Summary
Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. While canonical H3 is replenished during DNA replication in S-phase[4], loading of CENP-A in Drosophila and humans takes place in a replicationindependent manner from late mitosis to G15–9 This process requires the exchange or removal of so-called placeholder nucleosomes containing H3 and H3.3, which have been positioned on centromeric DNA-sequences during the previous S-phase[10,11]. Epitope-tag labeling of dCENP-A revealed that once fully incorporated, CENP-A turnover in healthy proliferating cells is almost exclusively restricted to replicative dilution[12,13] Some of this stability is conferred to CENP-A by other centromere factors that act on the intact DNA-bound nucleosome itself. CENP-C reshapes and clamps down the CENP-A nucleosome, CENP-N helps fastening CENP-A to the underlying DNA14,15
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