Double-strand DNA breaks recruit the centromeric histone CENP-A

Abstract

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

Fig. 1.

Endogenous CENP-A localizes to sites of laser-induced damage, along with DNA repair markers phosphorylated H2AX, Chk2, Nbs1, and 53BP1. (A) (Top left) Phase contrast image of human osteosarcoma (143b) cells just before laser targeting along a line (red). (Other panels) Matched confocal immunofluorescent imaging 90 min (at 25 °C) after laser targeting. (Red) Phospho-histone H2AX; (green) Thr-68 phospho-Chk2; (blue) DNA detected with DAPI; (grayscale) endogenous CENP-A signal. Note the small foci in the CENP-A image are centromeres. Note that the cells are motile: the orientation of cell #2 changed in the 90 min after targeting. (B) HeLa cell 90 min. (at 25 °C) after laser targeting along a single line. (Blue) DNA, (red) 53BP1 or Nbs1 phospho-Ser-343, and (green) CENP-A. Other foci in the CENP-A image are centromeres.

Fig. 2.

Rapid GFP-CENP-A accumulation at sites of DNA damage. (A–F) GFP-CENP-A cells before and after laser targeting at 25 °C. Phase contrast (A) before and (D) 5 min. after targeting the areas boxed in red. Epifluorescence images of GFP-CENP-A, immediately (B) before and (C) 4 or (E) 5 min. after initiating laser exposure. In most cells (numbered in white), GFP-CENP-A accumulated at the sites of targeting. [Cells #7, 8 and 10 (numbered in yellow) were bleached during laser targeting,] (F) Within 85 min, GFP-CENP-A formed foci within targeted regions. (G) Laser targeting as in (A–F), maintained at 37 °C after targeting: a CENP-A focus appears within ≈5 min after laser exposure, reaches its peak intensity ≈1 h after laser exposure, and then disappears approximately 15 min later. Timestamp represents hours:minutes:seconds.

Fig. 3.

Rapid GFP-CENP-A accumulation at double-strand breaks induced by I-SceI cleavage in human and mouse cells. (A) Mouse cells carrying an I-SceI target site co-integrated with lacO repeats (30) after transient transfection to express (green) GFP-mCENP-A and (red) mCherry-lacR and (top) without or (bottom) with HA-tagged I-SceI. (Blue) γ-H2AX; (grayscale) DNA detected with DAPI. (B) The same mouse cell line as in (A), transiently co-transfected to express (green) GFP-mCENP-A, (red) CFP-lacR and (grayscale) RFP-I-SceI-GR and (top) without TA or (bottom) after TA addition for 1 h. (Blue) γ-H2AX. (C–E) Human cells carrying a single SceI target site on chromosome 10 and multiple SceI target sites on chromosome 6 were transiently co-transfected to express GFP-CENP-A and RFP-I-SceI-GR. (C and D) Cells imaged for (red) RFP-SceI-GR or (green) GFP-hCENP-A and (C) without or (D) 1 h. after addition of TA, which induces nuclear accumulation of RFP-I-SceI-GR to cleave the DNA. (E) Timelapse images of a single cell transfected as in (C and D) immediately before and after addition of TA. Timestamp is minutes:seconds.

Fig. 4.

Histone H2B never accumulates in areas of laser-induced DNA damage. (A–D) Epifluorescence images of HeLa cells stably expressing YFP-H2B (A) before and (B–D) after laser exposure (red lines). (C) γ-H2AX and (D) YFP-H2B 90 min. after laser exposure. (E) Phase contrast image of HeLa cells stably expressing YFP-H2B before laser targeting. (F–J) YFP-H2B epifluorescence of the cells in (E) and (F and G) before or (H–J) after laser targeting. Red squares in (G and H) denote laser targeted areas. Timestamp is hours:minutes:seconds.

Fig. 5.

CENP-A recruitment to sites of DNA damage requires its centromere-targeting domain (CATD) and recruits centromeric nucleosome-associated factors CENP-N and CENP-U. (A) Schematic of CENP-A/H3 chimeric proteins and frequencies at which each is recruited to DNA damage foci. (B–E) Human HCT116 cells were transiently transfected. DNA damage was induced by laser targeting, and frequencies of focal accumulation at site of DNA damage were measured. (B) GFP-H3^CATD. (C) GFP-CENP-N. (D) GFP-CENP-U. (E) GFP-CENP-T.

Fig. 6.

CENP-A in function in DNA repair independent of H2AX, and possible models for CENP-A assembly at sites of DNA repair. (A) HCT116 cells generated by targeted disruption of NHEJ genes were transiently transfected to express GFP-CENP-A; frequencies of focus formation were measured after laser exposure. [WT (n = 47 cells); DNA-PKcs^−/− (n = 39 cells); LigIV^−/− (n = 57 cells); Ku86^+/− (n = 38 cells); P = 0.0202 (one-way ANOVA)]. (B) Frequencies of GFP-CENP-A recruitment to sites of laser-induced DNA damage in Ligase IV^−/− cells transfected to express mCherry-LigaseIV or mCherry-LigaseIV-R278H. (C) Percentages of cell death following 0 Gy, 2 Gy, and 10 Gy irradiation (done in triplicate) in cells with a stably integrated, GFP-CENP-A gene, either with or without tetracyline-mediated gene induction. Cell death was scored by small, bright nuclei [using Hoechst] and quantified 24 h later by automated microscopy (n = 2,500–5,000 cells per sample). Error bars show the standard devia tion. (D) Cell survival in a colony formation assay after 8 Gy irradiation of parental 293 cells or inducible GFP-CENP-A cells. (E) Frequency of (transiently transfected) GFP-mCENP-A recruitment to sites of laser-induced DNA damage in wild-type and H2AX null MEFs. (F) GFP-mCENP-A recruitment to focal DNA damage (red box) in an H2AX null cell. (G) Model for CENP-A recruitment to sites of DNA damage. Laser-mediated damage or Sce1 cleavage creates DNA double-strand breaks. CENP-A, CENP-N and CENP-U are recruited within 1–2 min. Three possible modes of CENP-A binding are shown (see Discussion).