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. 2013 Apr;33(7):1410-6.
doi: 10.1128/MCB.01654-12. Epub 2013 Jan 28.

γH2A-binding protein Brc1 affects centromere function in fission yeast

Si Young Lee  1 , Sophie Rozenzhak, Paul Russell
Affiliations

γH2A-binding protein Brc1 affects centromere function in fission yeast

Si Young Lee et al. Mol Cell Biol. 2013 Apr.

Abstract

The coordinated replication and transcription of pericentromeric repeats enable RNA interference (RNAi)-mediated transmission of pericentromeric heterochromatin in fission yeast, which is essential for the proper function of centromeres. Rad3/ATR kinase phosphorylates histone H2A on serine-128/-129 to create γH2A in pericentromeric heterochromatin during S phase, which recruits Brc1 through its breast cancer gene 1 protein (BRCA1) C-terminal (BRCT) domains. Brc1 prevents the collapse of stalled replication forks; however, it is unknown whether this activity influences centromere function. Here, we show that Brc1 localizes in pericentromeric heterochromatin during S phase, where it enhances Clr4/Suv39-mediated H3 lysine-9 dimethylation (H3K9me2) and gene silencing. Loss of Brc1 increases sensitivity to the microtubule-destabilizing drug thiabendazole (TBZ) and increases chromosome missegregation in the presence of TBZ. Brc1 retains significant function even when it cannot bind γH2A. However, elimination of the serine-121 site on histone H2A, a target of Bub1 spindle assembly checkpoint kinase, sensitizes γH2A-deficient and brc1Δ cells to replication stress and microtubule destabilization. Collective results suggest that Brc1-mediated stabilization of stalled replication forks is necessary for fully efficient transmission of pericentromeric heterochromatin, which is required for accurate chromosome segregation during mitosis.

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Figures

Fig 1
Fig 1
HU treatment increases Clr4-dependent Brc1 enrichment at centromeres. ChIP analysis of Brc1-GFP in log-phase cells (A) and cells treated with 12 mM HU for 4 h (B). Enrichment was calculated relative to the act1 (actin) locus (see Materials and Methods).
Fig 2
Fig 2
Brc1 enhances gene silencing in pericentromeric heterochromatin. (A) Diagram of centromere I (cen1) with ura4+ insertion sites (top panel). Gene-silencing assays in nonselective (N/S), selective (−URA), and counterselective (5-FOA) media (bottom panels). (B) qRT-PCR analysis of ura4 (otr::ura4+) transcript levels relative to a control transcript, act1+, normalized to the wild type.
Fig 3
Fig 3
Brc1 assists heterochromatin-specific modification of the histone H3 tail at the centromere. ChIP analysis using anti-dimethyl-histone H3K9 (H3K9me2) antibodies at cen(dg) relative to act1+, normalized to the wild type. Cells were either untreated or exposed to 12 mM HU for 4 h.
Fig 4
Fig 4
TBZ sensitivity in brc1Δ cells. (A) TBZ sensitivity caused by brc1Δ is additive to that caused by swi6Δ. Tenfold serial dilutions were incubated at 30°C for 2 days. (B) Mitotic abnormalities in brc1Δ cells released from a cdc25-22 arrest in the absence or presence of 5 μg/ml TBZ. Micrographs were taken at 60 min after release from the arrest. DNA was stained with DAPI and antitubulin antibody (TAT1). The arrow points to a lagging chromosome (or chromosome fragment) in brc1Δ cells treated with TBZ.
Fig 5
Fig 5
Loss of γH2A or Brc1 increases TBZ sensitivity in cells lacking the serine-121 site in histone H2A that is phosphorylated by Bub1. (A) Histone H2A mutants lacking the γH2A site (htaAQ [hta1-S129A hta2-S128A]) or the Bub1 phosphorylation site (htaSA [hta1-S121A hta2-S121A]) alone, or in combination (htaSA.AQ), or with brc1Δ were tested with TBZ or the indicated genotoxins. Cells were incubated at 30°C for 2 days. (B) Phosphorylation of histone H2A at serine-121 is not required for localization of Brc1 at centromeres. ChIP analysis of Brc1-GFP in cells treated with 12 mM HU for 4 h. Enrichment was calculated relative to the act1 locus (see Materials and Methods).
Fig 6
Fig 6
Model for Brc1 stabilizing replication forks in pericentromeric heterochromatin. Collisions between the replisome and RNA Pol II trigger γH2A formation by Rad3 (not shown), resulting in recruitment of Brc1. Stabilization of the replication fork by Brc1 favors RNAi-mediated disassociation of RNA Pol II from the chromosome, allowing fork restart by the Rik1-associated replisome (22). Loss of Brc1 destabilizes the fork and increases the frequency of HDR-mediated fork restart by a reassembled replisome that is not associated with the Rik1 complex. See the text for additional description.
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