Abstract
The fission yeast pericentromere comprises repetitive sequence elements packaged into heterchromatin marked by histone H3K9 methylation and Swi6 binding. Transient disruption of Swi6 during S phase allows a period of RNA synthesis which programs the RNAi machinery to maintain histone methylation. However, Swi6 is also required for early replication timing. We show that not only Swi6 but also the chromodomain protein Chp1 are delocalized during S phase. Different from loss of swi6, mutations that disrupt histone methylation in the centromere, chp1Δ and clr4Δ, undergo early DNA replication. However, timing is modestly delayed in RNAi mutants dcr1Δ or rdp1Δ, while hrr1Δ mutants resemble swi6Δ in their replication delay. Finally, we show that recruitment of RNA polymerase II in the centromere occurs independently of replication. These different effects indicate that replication timing is not simply linked to histone methylation.
Highlights
In eukaryotic cells, replication origins fire at different times in S phase depending on their position and local chromatin structure, and availability of limiting replication factors
Consistent with the early replication observed in swi6D clr4D double mutants [4], we find that clr4D as well as chp1D single mutants replicate early, indicating that the pericentromeric domains are intrinsically early-replicating in the absence of the RNA-inducedtranscriptional-gene-silencing complex (RITS) complex or histone methylation
Swi6 and Chp1 both bind to methylated histone H3K9 via their chromodomain [24]
Summary
Replication origins fire at different times in S phase depending on their position and local chromatin structure, and availability of limiting replication factors (reviewed in [1,2,3]). In S. pombe, the repetitive pericentromeric heterochromatin replicates early, in contrast to telomeres [4,5,6]. All these domains are characterized by binding of the Heterochromatin Protein 1 (HP1) homologue Swi to methylated histone H3K9 [7]. The RNAs are amplified by Rdp1-dependent RNA Polymerase Complex (RDRC) to generate double stranded RNAs that are further processed to siRNAs by Dcr. The RNAs are amplified by Rdp1-dependent RNA Polymerase Complex (RDRC) to generate double stranded RNAs that are further processed to siRNAs by Dcr1 These RNAs together with Ago, Chp and Tas form a RNA-inducedtranscriptional-gene-silencing complex (RITS) that targets nascent repeat transcripts at the centromere. Recent studies show that Swi acts upstream of the RDRC, via interaction with the Ers1 [12,13,14]
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