Figure 5.

SETD2 Promotes HR through LEDGF/CtIP-Facilitated Resection

(A) Coimmunoprecipitation of LEDGF and H3K36me3 in the presence or absence of DNA damage. U2OS cells were left untreated (U) or treated with CPT (15 μM for 4 hr). Reciprocal co-IPs are shown.

(B) ChIP analysis of LEDGF binding to the DSB site before and after I-SceI induction in NT or SETD2 knockdown DR-GFP cells. siNT, control siRNA-treated cells before cut; siS, SETD2 siRNA-treated cells before cut; siNT+IScI, control siRNA-treated cells after cut; siS+IScI: SETD2 siRNA-treated cells after cut. Numbers were quantified from one ChIP-PCR experiment and show LEDGF-ChIP over input.

(C) Microirradiation showing CtIP recruitment to the damage site in U2OS cells treated with nontargeting control siRNA (NT) or SETD2 siRNAs (si#3 and si#5). Fluorescent images were acquired using a confocal microscope (Zeiss); scale bars, 20 μm.

(D) HR efficacy (as measured by GFP reporter assay as in Figure 2D) in cells transfected with control siRNA (NT) or CtIP siRNA (siCtIP) or both CtIP and SETD2 siRNA (siC+siS). Error bars show SEM from three independent experiments, ^∗∗p < 0.01, n.s. not significant.

(E) CtIP depletion impedes resection at an AsiSI induced DSB. DNA was extracted from 4OHT-treated or untreated DIvA cells, transfected with control or CtIP siRNA, as indicated. Resection was analyzed, using a protocol detailed in Zhou et al. (2014), at an AsiSI-induced DSB reported to be repaired by a RAD51-dependent pathway (DSB-II in Aymard et al., 2014). The mean and SEM (n = 4, technical replicate) of a representative experiment are presented.

(F) SETD2 depletion impedes resection at an AsiSI induced DSB. DNA was extracted from 4OHT-treated or untreated DIvA cells, transfected with control or SETD2 siRNA, as indicated. Same as in (E) except that cells were transfected with a siRNA directed against SETD2. The mean and SEM (n = 4, technical replicate) of a representative experiment are presented. See also Figure S5.