H2A.Z depletion impairs proliferation and viability but not DNA double-strand breaks repair in human immortalized and tumoral cell lines

Abstract

In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z.

Keywords: DNA repair; H2A.Z; NHEJ; chromatin; homologous recombination; p400.

Figure 1. Effect of H2A.Z depletion on DSB repair by NHEJ (A) Schematic representation of the NHEJ substrate present in the cell lines. The translation of the GFP gene is suppressed by an upstream, out of frame translation start site (KOZ ATG), the cassette is flanked with 2 I-SceI sites. I-SceI expression induces the release of the Koz ATG and religation of DNA ends allows translation and expression of GFP (B) GCS5 and (C) GCV6 cells were transfected with siRNA (10 nM) then, 24 h later, transfected with I-SceI coding plasmid to induce DSB. Treatment with Nu7441 was performed at 5 µM during 48 h at the time of I-SceI transfection. NHEJ events were counted 48 h later by FACS analysis of GFP-positive cells. Results are the mean +/− sd from 3–5 independent experiments. Western blots monitoring the indicated proteins expression are also shown.

Figure 2. Effect of H2A.Z depletion on homology-directed repair of DSB. (A) Schematic representation of the subtrate present in the cells to measure DSB repair mediated by homologous recombination. Two inactive copies of GFP gene are present: one contains the cleavage site for I-SceI; the other one is truncated at the 5′ and 3′ ends. After cleavage intra- or interchromatid use of the truncated form of GFP to repair DSB generates functional GFP (B) RG37 cells were transfected with the different siRNA (10 nM), then, 24 h later, transfected with I-SceI plasmid to induce DSB. HDR efficiency was evaluated 48 h later by FACS analysis. Results are the mean +/− SD from 4 independent experiments. Western blots monitoring the indicated proteins expression are also shown (western blot monitoring the effects of p400 depletion are shown in ref. 17).

Figure 3. Clonogenic survival of IR exposed cells after H2A.Z depletion. U2OS cells transfected with the different siRNA were (A) plated to evaluate clonogenic efficiency or (C) exposed to ionizing radiations 24 h later and clonogenic assay performed. The same protocol was applied with the RG37 cells (B) to evaluate clonogenic efficiency and (D) to examine cell survival after ionizing radiations exposure. Colonies were revealed 10 d later by staining the plates with crystal violet. Results are the mean+/− sd of triplicate.

Figure 4. Effect of H2A.Z depletion on cell cycle distribution. Cell cycle distribution was examined by flow cytometry in U2OS (A) and RG37 (B) cells 48 h after siRNA transfection and staining with propidium iodide. Results are the mean +/− sd from 3 independent experiments.

Figure 5. Analysis of H2A.Z recruitment on DNA damage in living cells. Nucleus of U2OS cells transfected with 53BP1-GFP (A) or H2A.Z-GFP (B) plasmids were irradiated with pulsed laser (532 nm) to induce DNA damage then images were collected sequentially. (C) Nucleus of U2OS cells first transfected with siCtrl or sip400 then transfected with 53BP1-GFP plasmid and irradiated to induce DNA damage as in (A). Time after irradiation is indicated above the photographs.

Figure 6. Chromatin immunoprecipitation of H2A.Z at DNA double-strand break. ChIP experiment showing H2A.Z and gH2AX accumulation at DSB in AsiSI-ER-U2OS cells.