Control of sister chromatid recombination by histone H2AX - PubMed (original) (raw)

Control of sister chromatid recombination by histone H2AX

Anyong Xie et al. Mol Cell. 2004.

Abstract

Histone H2AX has a role in suppressing genomic instability and cancer. However, the mechanisms by which it performs these functions are poorly understood. After DNA breakage, H2AX is phosphorylated on serine 139 in chromatin near the break. We show here that H2AX serine 139 enforces efficient homologous recombinational repair of a chromosomal double-strand break (DSB) by using the sister chromatid as a template. BRCA1, Rad51, and CHK2 contribute to recombinational repair, in part independently of H2AX. H2AX(-/-) cells show increased use of single-strand annealing, an error-prone deletional mechanism of DSB repair. Therefore, the chromatin response around a chromosomal DSB, in which H2AX serine 139 phosphorylation plays a central role, "shapes" the repair process in favor of potentially error-free interchromatid homologous recombination at the expense of error-prone repair. H2AX phosphorylation may help set up a favorable disposition between sister chromatids.

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Figures

Figure 1

Figure 1. Impaired Gene Conversion of a Chromosomal DSB in _H2AX_−/− ES Cells

(A) HR-GFP reporter, filled arrows show promoters; open-headed arrow indicates I-SceI-induced HR, leading to alternative STGC or SCR/LTGC outcomes shown. (B) SCR reporter, ellipses “A” and “B” indicate artificial BsdR exons. (C) I-SceI induction of HR in HR-GFP reporter. Bars represent mean of four independent experiments, each with triplicates. Error bars indicate SEM. Paired t test _H2AX_−/− versus other groups, p < 0.001%. (D) Similar experiments in SCR reporter. Paired t test _H2AX_−/− versus other samples, p < 0.001%. (E) I-SceI-induced HR in isogenic ES cell clones containing HR-GFP or SCR reporters. Dots represent mean of triplicates in one experiment for each clone. Error bars indicate SEM. Unpaired t test (unknown variance) _H2AX_−/− versus other samples, p < 0.0001%. (F) I-SceI-induced chromosomal DSBs in ES cell lines transfected with I-SceI plasmid or vector control and harvested at times shown after transfection. Southern blotting was performed on BglII-digested gDNA with a GFP probe. Schematic of the SCR reporter with BglII and I-SceI sites is shown in upper section. Right, control BglII (“B”) and BglII/I-SceI (“B/S”) digests of H2AX+/+ gDNA.

Figure 2

Figure 2. Serine 139 Is Essential for H2AX HR Function

(A and B) I-SceI-induced GFP+ frequencies in _H2AX_−/− reporter cell lines HR-GFP (A) or SCR (B), stably expressing H2AX alleles indicated. Bars represent mean of four independent experiments, each with triplicates. Error bars indicate SEM. Paired t test between wt and other samples, p < 0.0001%; between SD and SA in (B): p < 0.001%. (C) Steady state levels of HA-H2AX protein in ES cell lines shown in (A) and (B). (D) I-SceI-induced GFP+ frequencies in ES cells transiently transfected with H2AX expression plasmids. Bars represent mean of two independent experiments, each with triplicates. Error bars indicate SEM. Paired t test between wt and any other samples in _H2AX_−/−, p < 0.0001%; in H2AX+/−, p < 0.005%; in H2AX+/+, not significant. (E) H2AX protein levels after transient transfection of H2AX alleles in ES cells containing SCR reporter—same experiment as (D). HA-H2AX migrates slower than endogenous H2AX. Antibody specific to wt unphosphorylated H2AX C-terminal tail binds poorly to S139 mutant proteins (middle row and data not shown); this accounts for the discrepancy between anti-HA and anti-H2AX signal seen here.

Figure 3

Figure 3. H2AX Regulates SCR

(A and D) I-SceI-induced GFP+ frequencies in ES cell lines containing the SCR reporter (A) or _H2AX_−/− ES cell lines containing the SCR reporter, stably reconstituted with H2AX alleles (D). These figures reproduce the data shown in Figures 1D and 2B. Bars represent mean of four independent experiments, each with triplicates. Error bars indicate SEM. (B and E) I-SceI-induced BsdR+ frequencies in ES cell lines in the same experiments depicted in (A) and (D). Bars represent mean of four independent experiments, each with triplicates. Error bars indicate SEM. Paired t test between _H2AX_−/− cells and others in (B), p < 0.001%. Paired t test between wt and any other samples in (D), p < 0.001%. Frequency of spontaneously arising BsdR+ cells was ∼0.001% in all cases and is not shown. (C and F) Ratios of I-SceI-induced BsdR+ to GFP+ frequency from the same experiments shown in (A) and (B) or (D) and (E). Bars represent mean of ratios in four independent experiments, each with triplicates. Error bars indicate SEM. All differences, not significant. (G) Southern blot analysis of I-SceI-induced BsdR+ colonies (BglII digests of gDNA, GFP probe). Cartoon shows schematic of unre-arranged reporter and of GFP triplication outcome. Lanes 1–3 and 6–8 are representative of GFP triplication events (H2AX status as indicated). Lanes 4 and 9 are representative of mixed clones with GFP triplication and parental reporter (compare Puget et al., 2004). Lanes 5 and 10 are representative of aberrant SCR/LTGC events. P, parental (unrearranged) reporter.

Figure 4

Figure 4. H2AX Suppresses SSA

(A) The SCR reporter and its SSA product after DSBR. (B) Southern blot analysis of I-SceI-trans-fected ES cell/SCR reporter clones that have lost the I-SceI site (Lanes 1–8, BglII digests of gDNA; GFP probe). P, parental (unrearranged) reporter. (C) Southern blot analysis of unselected bulk cultures 5 days after transfection of I-SceI (+) or control (−) plasmid (BglII digested gDNA; GFP probe). “C” shows migration of sequence-defined SSA product. Numbers below gel indicate proportion of 2.9 kb SSA product in lane, quantified by densitometry, corrected for transfection efficiency and GFP copy number.

Figure 5

Figure 5. BRCA1, Rad51, and CHK2 Act Independently of H2AX to Control HR

(A) The percentage of I-SceI-induced GFP+ cells from ES cell lines transfected with RNAi duplexes. H2AX+/+ or _H2AX_−/− ES cell lines, as indicated, were transiently transfected with control RNAi or RNAi against BRCA1, CHK2, or Rad51, together with I-SceI plasmid. GFP+ cells were quantified by FACS analysis 3 days posttransfection. Bars represent the average from three independent experiments, each with triplicates. Error bars indicate SEM. (B) Depletion of proteins in ES cells by corresponding RNAi. ES cells, as indicated, were transfected with RNAi and lysed 3 days posttransfection. Whole-cell extracts were resolved by SDS-PAGE and analyzed by Western blotting by using antibody against target proteins (BRCA1, CHK2, and Rad51) or α-actin as a loading control.

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