Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining - PubMed (original) (raw)

Methylation of histone H3 lysine 36 enhances DNA repair by nonhomologous end-joining

Sheema Fnu et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Given its significant role in the maintenance of genomic stability, histone methylation has been postulated to regulate DNA repair. Histone methylation mediates localization of 53BP1 to a DNA double-strand break (DSB) during homologous recombination repair, but a role in DSB repair by nonhomologous end-joining (NHEJ) has not been defined. By screening for histone methylation after DSB induction by ionizing radiation we found that generation of dimethyl histone H3 lysine 36 (H3K36me2) was the major event. Using a novel human cell system that rapidly generates a single defined DSB in the vast majority of cells, we found that the DNA repair protein Metnase (also SETMAR), which has a SET histone methylase domain, localized to an induced DSB and directly mediated the formation of H3K36me2 near the induced DSB. This dimethylation of H3K36 improved the association of early DNA repair components, including NBS1 and Ku70, with the induced DSB, and enhanced DSB repair. In addition, expression of JHDM1a (an H3K36me2 demethylase) or histone H3 in which K36 was mutated to A36 or R36 to prevent H3K36me2 formation decreased the association of early NHEJ repair components with an induced DSB and decreased DSB repair. Thus, these experiments define a histone methylation event that enhances DNA DSB repair by NHEJ.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Metnase dimethylates H3K36 at DSBs. (A) Western blot of H3K36me2 after γ-radiation. (B) Quantitative real-time PCR analysis using PCR primers spanning the I-SceI site. Plotted are relative values calculated as the inverse of the total amount of amplified puro DNA compared to input GAPDH DNA for I-SceI adenovirus MOIs of 100 and 1,000. (C) Schematic of the HT1904 I-SceI DSB cell system (see also

Figs. S3

and

S4

). (D) ChIP time course of methylated H3 species adjacent to a single induced DSB quantified by real-time PCR. Methylated H3K36 was not detected prior to DSB induction. For all ChIP and DSB repair experiments, each time point is the average of three quantitative real-time PCR measurements normalized to input DNA. All H3K36me2 ChIP data were also normalized to the presence of total H3 assessed by ChIP at the DSB. All data points include errors bars (SEM), but in many cases the error bars are smaller than data point symbols. Statistics were calculated for each time point vs. controls. In this and all subsequent figures, * indicates P ≤ 0.05 and ** indicates P ≤ 0.01. (E and F) ChIP analysis of Metnase and H3K36me2 adjacent to a single induced DSB. pcDNA-Metnase indicates Metnase overexpression, U6-si-Metnase indicates repression; pcDNA and U6 are empty vector controls. There was no detectable Metnase or H3K36me2 prior to DSB induction. (G) The Metnase D248S SET domain mutant prevents H3K36me2 formation at the DSB.

Fig. 2.

Fig. 2.

Coimmunoprecipitation of DNA repair components with H3K36me2 after IR. (A) Immunoprecipitates (IP) of H3K36me2 were collected from HT1904 cells before (C) and 15 or 60 min after 10 Gy γ-radiation and analyzed by Western blot for NBS1, Ku70, DNA ligase IV; input, nonspecific IgG, and H3K36me2 controls are also shown. ChIP analysis of early DNA repair components at a single induced DSB. Increasing Metnase levels enhances the presence of Ku70 (B) and phospho-NBS1 (C) at the single DSB, while repressing Metnase reduced the presence of these proteins. Neither Ku70 nor phospho-NBS1 were detected at the target DSB site prior to DSB induction (B and C).

Fig. 3.

Fig. 3.

Dimethylation of H3K36 enhances DSB repair. (A) Repair of a single DSB measured by real-time quantitative PCR in cells over- or underexpressing Metnase; values are inverse percentages of input (uncut) DNA prior to DSB induction and normalized to GAPDH (input) controls. Statistics calculated at each time point for over- or underexpression vs. cognate empty vector controls. (B) As in A but with cells expressing wild-type or D248S (SET-defective) Metnase. (C and D) Overexpression of JHDM1a demethylase reduces H3K36me2 presence at an induced DSB (C) and reduces DSB repair (D).

Fig. 4.

Fig. 4.

Expression of H3A36 and H3R36 mutant proteins limits Ku70 and NBS1 recruitment to DSBs and decreases DSB repair. (A) HT1904 cells transfected with vectors expressing WT H3, H3R36, or H3A36 were treated with 10 Gy IR or untreated and analyzed for H3K36me2 by Western blot. (B) Quantification of H3K36me2 signals in A normalized to actin loading controls. (C_–_E) ChIP analysis of Ku70 and NBS1, and DSB induction and repair in HT1904 cells expressing wild-type or mutant histone H3, or empty vector control.

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