Fig 2: Model of the function of LEDGF/p75 and HDGFRP2 in homologous recombination DNA repair. Repair of a DNA double strand break (DSB) by HR in active chromatin requires the histone H3K4me3- and H3K36me3-binding protein LEDGF/p75 to recruit RBBP8/CtIP and ensure efficient DNA end resection. The structurally highly similar protein HDGFRP2 binds preferentially to histone marks of silent genes (e.g. histone H3K9me3) and contributes, together with its interaction partner POGZ, to efficient DSB repair by HR. HDGFRP2 facilitates RBBP8/CtIP recruitment to the damage site and may play a role in the separate heterochromatic chromatin compartment or in chromatin silenced around the DNA DSB break. Its binding to silent chromatin may reflect its role e.g. in the inhibition of transcription and activation of Tip60 around the damage site.

Fig 3: Knockdown in HDGFRP2 inhibits DNA DSB repair by homologous recombination. (A) The homologous repair reporter contains an I-SceI restriction site, which upon I-SceI expression is cleaved to generate a DNA DSB. DSB repair by homologous recombination using truncated wild-type GFP (iGFP) as a template results in an intact GFP gene. (B) U2OS DR-GFP cells were transfected with control (C), RBBP8 or HDGFRP2 (H3 and H4) siRNAs for 48 h, followed by transfection with I-Sce-I and RFP (internal control) for 24 h. The efficiency of homologous recombination was determined by the GFP/RFP ratio analysed by flow cytometry. Representative immunoblots of indicated proteins from lysates of the same cells are shown on right. Error bars, SEM of four independent experiments. when compared with cells transfected with control siRNA in parallel. (C) Representative immunoblots of indicated proteins from lysates of U2OS cells transfected with control (C), HDGFRP2 (H3 and H4) or PSIP1 siRNAs for 72 h and left untreated or subjected to treatment with 1 μM camptothecin (CPT) for 1 h followed by 1 h recovery. The quantification of the indicated bands is presented below. The experiment was repeated at least four times with similar results. (D) Representative immunoblots of indicated proteins from lysates of U2OS cells transfected with control (C) or HDGFRP2 (H4) siRNAs and left untreated or subjected to 5 Gy ionizing radiation (IR) followed by 1, 2 or 4 h recovery. The experiment was repeated at least four times with similar results. Two experiments with H3 and H6 siRNAs gave similar results. (E) Representative immunoblots of indicated proteins from lysates of U2OS cells transfected with H4 siRNA-resistant mutant of HDGFRP2 (HDGFRP2*, +) or empty vector (−) together with control (C) or HDGFRP2 (H4) siRNAs for 72 h and left untreated or subjected to treatment with 1 μM camptothecin (CPT) for 1 h followed by 1 h recovery (left). The quantification of P-S4/S8-RPA2 relative to total RPA2 is shown on the right. Error bars, SD of three independent experiments. *P < 0.05, **P < 0.01 when compared with cells transfected with control siRNA in parallel (B) or as indicated (E).

Fig 4: IKZF1 regulates DNA end resection and HR. (A) Illustration of the design of Taqman qPCR primers and probes (black arrows) for measuring resection at sites adjacent to the AsiSI sites (red arrows). The primer pairs are across BsrGI restriction sites. (B) qChIP analysis of IKZF1 recruitment around sites of DSBs. NCI-H929 cells stably expressing HA-ER-AsiSI were cultured in the absence or presence of 0.5 µM 4-OHT. qChIP experiments were performed using anti-IKZF1 or anti-CtIP antibodies with primers that covered the DNA sequences flanking the AsiSI cutting site and the distal region of the break. CtIP was used as a positive control. (C-F) Analysis of HRR (C, D) and alt-EJ (E, F) using the DR-GFP and EJ2-GFP reporter assay in U2OS cells showed that HRR is promoted by IKZF1 overexpression. (G-H) RPMI-8226 cells stably expressing control shRNA and IKZF1 shRNA were subjected to CPT (100 nM) for 1 h, the RPA2 foci of which were then examined. Scale bar: 20 µm (G). The cellular extracts were prepared for western blotting with the indicated antibodies (H). Data are mean ± s.d. p-values were analyzed by one-way analysis of variance (ANOVA) and two-sided Student's t-test. *p < 0.05, ns: no significant.

Fig 5: HDGFRP2 associates with RBBP8/CtIP, heterochromatin-binding proteins and histone marks of transcriptionally silent chromatin. (A) Lysates of U2OS cells left untreated or treated with 1 μM camptothecin (CPT) for 1 h were immunoprecipitated with HDGFRP2 antibody (HDGFRP2) or IgG control and analysed by immunoblotting of indicated proteins. Similar results were obtained in a minimum of three independent experiments. (B–D) Co-localization of HDGFRP2 and RBBP8/CtiP was analysed using rabbit anti-HDGFRP2 and murine anti-RBBP8/CtiP antibodies and Duolink-based in situ proximity ligation assay (PLA) with appropriate secondary antibodies. U2OS cells were first transfected with control (C) or HDGFRP2 (H4) siRNA for 72 h and then left untreated or subjected to treatment with 1 μM camptothecin (CPT) for 1 h with or without a 4 h recovery. Representative confocal images of PLA-stained cells, in which co-localization of the antibodies used results in red (624 nm) fluorescence are shown in (B). Median values of nuclear PLA signals from a minimum of 75 (control siRNA) cells per condition presented as floating-bar plots are shown in (C) and immunoblot demonstrating the efficacy of H4 siRNA in (D). The specificity of the PLA signal was confirmed by the significant reduction of the signal by H4 siRNA. Note, that the remaining HDGFR2 protein in H4 siRNA-treated cells can also result in the PLA signal. DNA was stained with DAPI to visualize the nuclei. (E) Biotin-conjugated histone peptides were incubated with U2OS lysates, followed by a pulldown with streptavidin-coated magnetic beads. Representative immunoblot of indicated proteins. The vertical line indicates where the blot was cut to remove non-essential bands after staining and exposure. (F) Co-localization of HDGFRP2 and H3K9me3 was analysed using goat anti-HDGFRP2 and rabbit anti-H3K9me3 antibodies and Duolink-based in situ PLA with appropriate secondary antibodies in U2OS cells left untreated or treated with 1 μM camptothecin (CPT) for 1 h with or without 1 h recovery. Median values of nuclear PLA signals from a minimum of 75 cells per condition (right) are shown as floating-bar plots. DNA was stained with DAPI to visualize the nuclei. Representative confocal images are shown in Supplementary Figure S2. Scale bars, 10 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.