Genome-wide screen of human bromodomain-containing proteins identifies Cecr2 as a novel DNA damage response protein

Abstract

The formation of γ-H2AX foci after DNA double strand breaks (DSBs) is crucial for the cellular response to this lethal DNA damage. We previously have shown that BRG1, a chromatin remodeling enzyme, facilitates DSB repair by stimulating γ-H2AX formation, and this function of BRG1 requires the binding of BRGI to acetylated histone H3 on γ-H2AX-containing nucleosomes using its bromodomain (BRD), a protein module that specifically recognizes acetyl-Lys moieties. We also have shown that the BRD of BRG1, when ectopically expressed in cells, functions as a dominant negative inhibitor of the BRG1 activity to stimulate γ-H2AX and DSB repair. Here, we found that BRDs from a select group of proteins have no such activity, suggesting that the γ-H2AX inhibition activity of BRG1 BRD is specific. This finding led us to search for more BRDs that exhibit γ-H2AX inhibition activity in the hope of finding additional BRD-containing proteins involved in DNA damage responses. We screened a total of 52 individual BRDs present in 38 human BRD-containing proteins, comprising 93% of all human BRDs. We identified the BRD of cat eye syndrome chromosome region candidate 2 (Cecr2), which recently was shown to form a novel chromatin remodeling complex with unknown cellular functions, as having a strong γ-H2AX inhibition activity. This activity of Cecr2 BRD is specific because it depends on the chromatin binding affinity of Cecr2 BRD. Small interfering RNA knockdown experiments showed that Cecr2 is important for γ-H2AX formation and DSB repair. Therefore, our genomewide screen identifies Cecr2 as a novel DNA damage response protein.

Fig. 1

The inhibitory activity of BRG1 BRD for γ-H2AX formation is specific. (A) 293T cells were transfected with an empty vector or the indicated expression vectors and left untreated or irradiated by 10 Gy. Cells were collected after 1 h and divided into two for preparations of whole cell lysates and histone extracts, which were subjected to immunoblotting as indicated. The expression of α-tubulin and H2A was analyzed for loading control. (B) 293T cells were transfected with indicated expression vectors, irradiated by 5 Gy and fixed after 1 h for dual stain with anti-Myc and anti-γ-H2AX antibodies in the presence of DAPI. Representative confocal images, capturing both transfected and non-transfected cells, are shown. (C) The average number of γ-H2AX foci per cells was obtained by counting at least 50 cells, both non-transfected (control) and transfected, in each experiments of (B). Error bar indicates mean ± s. d. of three independent experiments. (D) After transfection with indicated vectors, 293T cells were left untreated (0 Gy) or irradiated by 1–5 Gy and subjected to colony forming assays. (E) 293T cells, after transfection with indicated vectors, were subjected to biochemical fractionation using detergent, and soluble (S, cytoplasmic and soluble nuclear proteins) and insoluble (I, chromatin-bound proteins) fractions were analyzed by immunoblotting. GAPDH and H2A were used as indicators of S and I fractions, respectively.

Fig. 2

Expression of 52 individual human BRDs in 293T cells. (A) The map of the plasmid vector expressing GFP-BRD is shown. Each of the 52 BRDs contains 10 flanking amino acids on either side and a NLS, nuclear localization signal. (B) 293T cells were transfected with vectors expressing GFP or each of the 52 GFP-BRD. Expression of the GFP proteins was analyzed by immunoblotting using anti-GFP and anti-α-tubulin (loading control) antibodies as indicated.

Fig. 3

Genome-wide screen for human BRDs having γ-H2AX inhibition activity. (A) 293T cells were transfected with vectors expressing the indicated GFP-BRDs, irradiated by 5 Gy and fixed after 1 h for dual stain with anti-γ-H2AX and anti-53BP1 antibodies before confocal images were captured. The average number of γ-H2AX and 53BP1 foci per cell was obtained by counting at least 200 GFP-positive cells, and data are presented as percentages relative to foci in cells expressing GFP only. Error bars indicate mean ± s.d. of three to six independent experiments. (B) Representative confocal images from the experiments described in (A) are shown.

Fig. 4

Cecr2 BRD specifically inhibits γ-H2AX formation and cell survival after DNA damage. (A) The 293T cells were transfected with indicated expression vectors and whole cell lysates were subjected to immunoblotting. (B) The 293T cells, transfected as described in (A), were subjected to colony forming assays after irradiation by various doses of IR. Error bars indicate mean ± s.d. of three independent experiments. (C) 293T cells, transfected with indicated expression vectors, were divided into two groups 1 h after irradiation by 5 Gy. One group was used to prepare whole cell lysates for immunoblotting. (D) The other group of cells from (C) were fixed and dually stained by anti-γ-H2AX and anti-53BP1 antibodies. The average number of γ-H2AX and 53BP1 foci per cell was obtained by counting at least 60 GFP-positive cells and depicted as a graph. Error bars indicate mean ± s.d. of three independent experiments. Control indicates cells expressing GFP only. (E) The 293T cells transfected with indicated expression vectors and subjected to colony forming assays after irradiation by various doses of IR. Error bars indicate mean ± s.d. of three independent experiments. (F) The 293T cells were transfected with indicated expression vectors and subjected to detergent-mediated fractionation as described in Fig. 1E. Soluble and insoluble fractions were analyzed by immunoblotting. Relative band intensity of Cecr2 as well as monomer and dimer Cecr2 BRDs in S and I fractions were determined by densitometer and shown below corresponding bands.

Fig. 5

Cecr2 is important for γ-H2AX formation and DSB repair. (A) The 293T cells were transfected with pSuper vectors expressing non-specific or three different Cecr2-specific siRNAs and divided into three groups. One group was used to analyze Cecr2 knockdown by immunoblotting. (B) The second group of cells was irradiated by 5 Gy and fixed for dual stain with anti-γ-H2AX and anti-53BP1 antibodies before confocal images were captured. The average number of γ-H2AX and 53BP1 foci per cell was obtained by counting at least 100 cells and depicted as a graph. Error bars indicate mean ± s.d. of three independent experiments. (C) Representative confocal images from the experiments in (B) are shown. (D) The last group of cells was irradiated by 20 Gy, collected immediately (0 h) or after various lengths of recovery time, and subjected to neutral comet assay to monitor progress of DSB repair. Average tail moments per cell, representing remaining unrepaired DSBs, were obtained by counting at least 300 comet images and depicted as graph. Error bars indicate mean ± s.d. of three independent experiments. (E) Representative comet images of the experiments in (C) are shown. (F) 293T cells were transfected as described in (A) and subjected to colony forming assays. Error bars indicate mean ± s.d. of three independent experiments.