Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity

Abstract

Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability as well as therapeutic targets in cancer. Here, we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using several cell-based assays and proteomic interaction network analysis. Applying these approaches, we identify 24 of the 42 BRD proteins as promoters of DNA repair and/or genome integrity. We identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allowing direct acetylation by PCAF, and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrids (R-loops) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on topoisomerase II, and BRD2 directly bound and activated topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a framework to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition.

Keywords: DNA damage response; DNA repair; R-loops; bromodomain; chromatin; homologous recombination.

Figure 1.

Human BRD proteins promote DNA double-strand break repair and genome stability. (A) Functional classification of human BRD proteins into eight subfamilies. DNA damage-associated BRD proteins indicated with red circle. (B) Comparative analysis of HR and NHEJ repair in cells deficient for each individual BRD protein. Individual BRD genes were depleted with siRNAs and analyzed for DSB repair in U2OS cells containing HR (DR) and NHEJ (EJ5) reporters. Data represent the mean of three independent experiments. (C) Ionizing radiation sensitivity screen for BRD proteins. Clonogenic survival assays were performed on siRNA-depleted BRD proteins that scored HR-deficient in B. Data shown is the ratio of relative survival for 8 Gy from three biologically independent experiments and represent the mean ± SEM. Results were normalized to control, nontargeting siRNA (siCtrl) treated cells and <75% of control values are indicated in red. (D) Endogenous DNA damage screen for BRD proteins. BRD proteins were depleted by siRNAs as in C and analyzed by immunofluorescence for the DNA damage marker γH2AX. BRD-deficient cells exhibiting an increase of γH2AX foci >4 standard deviations of siCtrl (4 SDs) are labeled in red. Data represent mean ± SEM from >100 cells. (E) BRD-deficient cells exhibit chromosome mis-segregation. Individual BRD proteins were depleted as in B, and cells were analyzed for micronuclei formation (see Materials and methods). An increase of >4 SDs of siCtrl are marked in red. Data represent the mean ± SEM from >100 cells.

Figure 2.

Protein interactome network of human BRD proteins. (A) Schematic overview of approach to identify protein-protein interactome of BRD proteins involved in HR repair. (B) Network of HR-promoting BRD proteins. Green hexagon-shaped nodes correspond to BRD-baits and blue circles indicate prey proteins. Gray lines indicate bait-prey interactions and green lines indicate BRD-BRD protein interactions. Green circles indicate BRD proteins (prey) identified as bait-prey interactions. (C,D) Subnetworks of GCN5/PCAF HAT-BRD and BET proteins. BRD bait and prey proteins are indicated as in B. For prey protein interactions, gene names are provided and placed into several categories as described in the legends. SAGA complex components are based on CORUM (Giurgiu et al. 2019) complex ID 6643 and ComplexPortal (Meldal et al. 2015) complexes CPX-900 and CPX-656. Individual BET BRD protein interactomes are shaded (BRD2: green; BRD4: purple; BRD3: pink).

Figure 3.

PCAF is a DNA damage response factor. (A) Knockout (KO) of PCAF by CRISPR/Cas9 in U2OS cells (left panel) and IR-sensitivity analyses by clonogenic assay (right panel). Knockout of PCAF was confirmed by western blotting with a PCAF-specific antibody. For IR sensitivity, colonies from undamaged and IR-damaged cells were counted, normalized to undamaged controls, and values were plotted as percent survival. Data represent the mean ± SEM; N = 3. (B) Loss of PCAF results in increased DSBs following ionizing radiation (IR) as detected by neutral comet assay (left panel, quantified in right panel). For all box-and-whisker plots, the box depicts 25%–75%, whiskers are 10%–90%, and the median is indicated. Data represent the mean ± SEM from >100 cells. (***) P < 0.001. (C) CRISPR-mClover HR assay (left) and random plasmid integration NHEJ assay (right). mClover-HR donor vector was transfected with Cas9-gRNA into U2OS WT or PCAF KO cells. Percentage of mClover-positive cells was normalized to control cells. NHEJ repair efficiency was analyzed by random plasmid integration assay with normalization to control cells. Data represent the mean ± SD; N = 3. (**) P < 0.01, (***) P < 0.001. (D) GFP-PCAF translocates to laser-induced DNA damage sites. (E) Schematic illustration of PCAF mutants. (F) C-terminal region (containing BRD domain) of PCAF promotes recruitment to DNA lesions. GFP-tagged PCAF mutants were monitored (left panel) and quantified (right panel) by live cell imaging using confocal microscopy. (G) C-terminal region (containing BRD domain) of PCAF is required for efficient chromatin binding. U2OS cells were fractionated following IR (10 Gy) treatment and analyzed by western blotting with indicated antibodies. (H) PCAF binds to acetylated histone 4 (H4Ac) via its C-terminal region (containing the BRD domain). A modified histone peptide array was performed with recombinant PCAF WT and C-terminal deletion mutant (upper panel). Lower black box shows a 2× magnification of original images with highly bound peptides indicated. (I) The C-term-containing BRD region of PCAF binds to H4ac. Biotinylated H4 peptides were incubated with GFP-PCAF WT and mutant overexpressed HEK-293 cell extracts and then immunoprecipitated with anti-GFP antibody. (J) Recruitment of PCAF to DNA lesions requires Tip60. GFP-tagged PCAF was monitored (left panel) and quantified (right panel) in siCtrl and siTip60 cells as in Figure 3F. For laser microirradiation experiments in D, F, and J, white dotted lines indicate laser paths and all images were normalized to undamaged regions. Data represent the mean ± SEM from >10 cells.

Figure 4.

PCAF regulates H2BK120 ubiquitylation and acetylation to promote DSB repair. (A) PCAF protein interactome network, including the SAGA DUB module (see Fig. 2C). (B) PCAF interacts with the SAGA DUB module. Cell extracts from SFB-PCAF expressing HEK-293 cells were immunoprecipitated with streptavidin beads and analyzed by western blotting. (C) PCAF promotes DNA damage signaling following IR treatment. U2OS WT and PCAF KO cells were treated with IR, collected at the indicated times, and cell lysates analyzed by western blotting with the indicated antibodies. (D) H2BK120 ubiquitylation accumulates in PCAF KO cells after IR treatment. Samples were analyzed as in C. (E) PCAF promotes USP22 recruitment to DNA damage sites. GFP-tagged USP22 was monitored (left panel) and quantified (right panel) following laser microirradiation in U2OS WT and PCAF KO cell lines by confocal microscopy. White dotted lines indicate laser paths, and all images were normalized to undamaged regions. Data represent the mean ± SEM from >10 cells. (F) N-AT and C-terminal BRD domains promote H2BK120 deubiquitylation. PCAF KO cells were transfected with SFB-tagged PCAF WT and derivatives followed by western blot analysis as in C. (G) PCAF N-AT and C-terminal domains facilitate HR. HR was measured by Cas9/EGFP-LNMB1 HR assay. Data represent the mean ± SEM; N = 3. (**) P < 0.01, (***) P < 0.001, (n.s.) not significant. (H) Inhibition or depletion of PCAF sensitive to PARP inhibitor. Cells were treated with olaparib and GSK4027 as indicated. Cell survival was analyzed by clonogenic assays. Data represent the mean ± SEM; N = 3. (I) PCAF bromodomain inhibitor (GSK4027) suppresses PCAF and USP22 recruitment to DNA damage sites. GFP-tagged PCAF and USP22 were monitored (left panel) and quantified (right panel) following laser microirradiation in DMSO- and GSK4027-treated cells by confocal microscopy. White dotted lines indicate laser paths, and all images were normalized to undamaged regions. Data represent the mean ± SEM from >10 cells. (J) Recombinant PCAF acetylates H2BK120. An acetylation assay was performed with purified PCAF WT and H2B. H2BK120 acetylation was detected using a specific antibody. (K,L) PCAF but not GCN5 is required for H2BK120ac at DSBs. Site-specific DSBs in DlvA cells were analyzed by ChIP assays using the indicated primers. DSB I and II represent HR-prone DSB sites. Data represent the mean ± SEM; N = 3. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (n.s.) not significant. (M) H2BK120 ubiquitylation to acetylation switch following DNA damage requires Tip60.

Figure 5.

RNA-DNA hybrid-induced DNA breaks form in BET-protein inhibited cells. (A–C) Inhibition of BET proteins trigger DNA breaks. Cells treated with the BET-BRD protein inhibitor (JQ1) were analyzed by western blotting with the DNA damage marker γH2AX. (B,C) Cells were treated with JQ1 (B) or indicated siRNAs (C); DNA breaks were detected by neutral comet assay (left panel) and tail moments were quantified (right panel). Data represent the mean ± SEM from >100 cells. (*) P < 0.05, (***) P < 0.001. (D) Transcription promotes DSBs in BET protein-deficient cells. JQ1 and/or transcription inhibitor (triptolide) were added to U2OS cells and γH2AX was analyzed by western blotting. (E) Inhibition of BET-proteins induced R-loops. Immunofluorescence (IF) analysis was performed with S9.6 antibody and mCherry-tagged RNaseH1. Nuclear S9.6 intensities were quantified with Image J (right panel). Diminution of nuclear S9.6 signal by mCherry-tagged RNaseH1 overexpression confirmed R-loops. (F) Quantification of R-loops by dot blot. Cells were treated with JQ1 or indicated siRNAs and purified genomic DNA ± RNaseH1 was analyzed with S9.6 antibody and α-ssDNA as a loading control (upper panel). The intensity of S9.6 was measured by Image J and normalized to DMSO or siCtrl (lower panel). Data = mean ± SEM; N = 3. (G) R-loops, and their associated DNA damage, that form in BET-protein inhibited cells require transcription. R-loop and γH2AX accumulation was monitored by IF in the presence or absence of triptolide, and images were analyzed as in E. (H) BET-inhibition induced R-loop generated DSBs in BET-inhibited cells. Inducible mCherry-RNaseH1 cell lines were treated with JQ1 with and without doxycycline (DOX) and compared to DMSO control cells. DSBs were detected by neutral comet assay (left panel, quantified in right panel). Data represent the mean ± SEM from >100 cells. (I,J) Resolution of R-loops by RNAseH1 suppresses DNA damage in BET-inhibited cells. DNA damage was monitored as in A in the presence or absence of RNaseH1 in JQ1 (I) or BET-BRD protein siRNA-treated cells (J). (K) BET inhibition-mediated R-loops occur in replicating (EDU+) and nonreplicating (EDU−) cells. R-loops were analyzed as in E. Representative images are shown (quantified in right panel). For the IF experiments in E–K, data represent the mean ± SEM from >100 cells. For all box-and-whisker plots, the box depicts 25%–75%, whiskers are 10%–90%, and median is indicated. (*) P < 0.05, (***) P < 0.001, (n.s.) not significant.

Figure 6.

BRD2 regulates topoisomerase I to prevent topoisomerase II-dependent DNA damage. (A) Network analysis of BET proteins interactomes. BET-BRD proteins interact with topoisomerase I (TOP1). (B) BRD2 protein interacts with TOP1. GFP-tagged BRD2 was transfected into U2OS cells and immunoprecipitated with anti-GFP. Samples were analyzed by western blotting with indicated antibodies. (C) BRD2 directly interacts with TOP1 in vitro. A protein interaction assay was performed by far western using recombinant BRD2 and TOP1. (D,E) BRD2 promotes TOP1 relaxation activity. TOP1 activity was measured by a plasmid relaxation assay. Relaxed and nicked DNA was monitored by agarose gel electrophoresis. (SC) Super-coil DNA, (R,N) relaxed and nicked DNA. Data represent the mean ± SEM; N = 3. (*) P < 0.05, (***) P < 0.001, (n.s.) not significant. (F) Schematic illustration of BRD2 mutants. (G) BRD2 promotes TOP1 relaxation activity via its C-terminal domain. TOP1 activity was measured by plasmid relaxation assay as in D,E. (H) TOP1 interacts with the C-terminal domain of BRD2. Cell extracts from GFP-BRD2 WT and mutants expressing HEK-293 cells were immunoprecipitated with GFP antibody and analyzed by western blotting. (I) Topoisomerase II (TOP2)-induced DSBs after JQ1 treatment. γH2AX foci were monitored by immunofluorescence (upper panel) and quantification of γH2AX foci per cell was plotted (lower panel). Data represent the mean ± SEM from >100 cells. (***) P < 0.001, (n.s.) not significant. For all box-and-whisker plots, the box depicts 25%–75%, whiskers are 10%–90%, and median is indicated. (J,K) Cells were cotransfected with indicated siRNAs and siTOP2. DNA damage production was measured by immunofluorescence (J) and western blotting (K) using a γH2AX antibody. The number of γH2AX foci per cell was quantified, and data represent the mean ± SEM from >100 cells in F. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (n.s.) not significant. For all box-and-whisker plots, the box depicts 25%–75%, whiskers are 10%–90%, and the median is indicated.

Figure 7.

Systematic analysis of BRD proteins in human cells reveals roles in maintaining genome integrity. (A) Interaction network of human BRD proteins involved in DNA repair. Human BRD proteins display high levels of connectivity and widespread involvement with DSB repair, especially HR. BRD bait proteins are shown in green hexagons; other BRD proteins in green rectangles. (B) Model of PCAF in DSB repair. PCAF translocation to DNA damage sites is reliant on H4 acetylation by Tip60. PCAF recruits the DUB USP22 to deubiquitylate H2BK120 and directly acetylate H2BK120. PCAF thus regulates the H2BK120 PTM switch from ubiquitin to acetylation at DNA damage sites. (C) Model for BET-BRD protein promotion of transcription that suppresses R-loop formation and genome instability via regulation of TOP1 activity. BRD2 and BRD4 promote TOP1 activity to inhibit R-loop formation during transcription. Depletion or inhibition of BRD2 and BRD4 induces R-loop accumulation and TOP2-dependent generation of DSBs, which may resolve R-loop/RNA polymerase II obstructions.