Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press RESOURCE/METHODOLOGY Systematic bromodomain protein screens identify homologous recombination and R-loop suppression pathways involved in genome integrity Jae Jin Kim,1,2,9 Seo Yun Lee,1,2,9 Fade Gong,1,2,8 Anna M. Battenhouse,1,2,3 Daniel R. Boutz,1,2,3 Aarti Bashyal,4 Samantha T. Refvik,1,2,5 Cheng-Ming Chiang,6 Blerta Xhemalce,1,2,7 Tanya T. Paull,1,2,5,7 Jennifer S. Brodbelt,4,7 Edward M. Marcotte,1,2,3 and Kyle M. Miller1,2,7 1Department of Molecular Biosciences, 2Institute for Cellular and Molecular Biology, 3Center for Systems and Synthetic Biology, 4Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, USA; 5The Howard Hughes Medical Institute; 6Simmons Comprehensive Cancer Center, Department of Biochemistry, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; 7Livestrong Cancer Institutes, Dell Medical School, The University of Texas at Austin, Austin, Texas 78712, USA 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 ge- nome 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 pro- teins, 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: bromodomain; chromatin; homologous recombination; DNA repair; DNA damage response; R-loops] Supplemental material is available for this article. Received July 28, 2019; revised version accepted October 28, 2019. Preserving the integrity of the genome is paramount for and Nussenzweig 2017; Xia et al. 2019), cells engage spe- maintaining cellular and organismal homeostasis. In eu- cialized signaling pathways termed the DNA damage re- karyotes, the nuclear genome is organized into chromatin, sponse (DDR) that detect, signal, and repair DNA which participates in compacting the genome and regulat- lesions (Jackson and Bartek 2009; Ciccia and Elledge ing its accessibility to promote cell identity and function. 2010). DNA double-strand breaks (DSBs) are a particularly Given the constant bombardment of DNA by exogenous deleterious form of DNA damage, which can provoke and endogenous factors including radiation, carcinogens, genome instability including insertions, deletions, trans- reactive oxygen species, replication stress, and dysregu- locations, and chromosome loss. DSBs are repaired by lated protein products (Jackson and Bartek 2009; Tubbs two main pathways in mammalian cells, homologous 8Present address: Department of Biochemistry and Molecular Biology, © 2019 Kim et al. This article is distributed exclusively by Cold Spring Baylor College of Medicine, Houston, TX 77030, USA Harbor Laboratory Press for the first six months after the full-issue publi- 9These authors contributed equally to this work. cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding author: [email protected] six months, it is available under a Creative Commons License (Attribu- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.331231. tion-NonCommercial 4.0 International), as described at http://creative- 119. commons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 33:1751–1774 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/19; www.genesdev.org 1751 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Kim et al. recombination (HR) and classical nonhomologous end- BRD proteins are highly mutated and/or aberrantly ex- joining (NHEJ). The importance of DDR pathways is high- pressed in cancer, which has motivated targeting BRD lighted by the various diseases associated with DDR proteins therapeutically (Muller et al. 2011; Barbieri defects, including neurodegenerative disorders, immune et al. 2013; Zhang et al. 2015). These efforts are highlight- deficiencies, and cancer (Jackson and Bartek 2009; Negrini ed by the development and use of BET inhibitors, includ- et al. 2010). ing JQ1 and I-BET (Filippakopoulos et al. 2010; Dawson DNA damage and the responses and activities elicited et al. 2011), which are widely used in preclinical and clin- by it are carried out within the context of chromatin. ical studies as therapeutic strategies to target various can- These chromatin-based DDR pathways ensure the coordi- cers (Boi et al. 2015; Andrieu et al. 2016). Given the dual nation of other DNA-templated processes such as tran- functions of BRD proteins in the DDR and cancer, under- scription and replication with the signaling and repair of standing mechanistically how BRD proteins promote ge- DNA damage, including DSBs (Lukas et al. 2011; Polo nome stability, a feature often lost in cancer (Negrini and Jackson 2011; Miller and Jackson 2012; Gong and Mil- et al. 2010), will be critical for revealing the involvement ler 2013; Jackson and Durocher 2013; Agarwal and Miller of BRD proteins in cancer as well as for guiding the use of 2016; Gong et al. 2016; Kim et al. 2019). For example, the cancer therapies targeting BRD proteins. As a means to ad- histone variant H2AX is phosphorylated by ATM and dress this question, we have systematically analyzed the DNA-PK at DSBs, which promotes the accumulation of involvement of all ubiquitously expressed human BRD DDR factors into microscopically visible foci at break proteins in promoting genome stability using diverse ex- sites including the DDR factors 53BP1 and BRCA1 (Polo perimental approaches. We find that the deficiency of and Jackson 2011; Scully and Xie 2013). 53BP1 is a multi- the majority of BRD proteins results in chromosome in- valent chromatin interacting protein that binds to ubiqui- stability, reduced DNA damage tolerance, and DNA tylated histone H2A and methylated H4, while BRCA1 DSB repair defects, especially in HR repair. These data interacts with the nucleosome to promote its ubiquityla- and our construction of BRD protein interactomes provid- tion activity on H2A (Fradet-Turcotte et al. 2013; Panier ed pivotal insights that led directly to the identification of and Boulton 2014; Densham and Morris 2017). Chromatin two new genome integrity pathways by BRD proteins de- plays an essential role in the DDR and in DSB signaling scribed here. We found that the HAT PCAF and an associ- and repair. ated DUB complex localize to DNA damage sites, where Acetylation represents a key posttranslational modifi- PCAF engages TIP60 acetylated histones with its BRD cation that regulates chromatin structure and function, to trigger a ubiquitin-acetylation switch on histone H2B including in the DDR (Gong et al. 2016; Fujisawa and Fil- that promotes HR repair. We further show that BRD2 or ippakopoulos 2017). Acetylation is catalyzed by histone BRD4 deficiency, either by depletion using siRNAs or tar- acetyltransferases (HATs) (Lee and Workman 2007; Ver- geting with the small molecule Pan-BET BRD inhibitor din and Ott 2015) and erased by histone deacetylases JQ1, results in aberrant transcription and control of topo- (HDACs), (Seto and Yoshida 2014), which dynamically logical topoisomerase enzymes and R-loop formation, re- regulate this mark on both histone and nonhistone pro- sulting in DSBs. Collectively, these data reveal BRD teins. Acetylated lysines are recognized by multiple recog- proteins as key chromatin reader proteins that are vital nition reader domains, including the bromodomain for maintaining the integrity of the genome through repair (BRD), which is found in 42 human proteins that play and transcriptional responses. key functions in chromatin regulation, including tran- scription and chromatin remodeling (Filippakopoulos and Knapp 2012; Filippakopoulos et al. 2012; Gong et al. Results 2016; Fujisawa and Filippakopoulos 2017). Over one-third Systematic identification of bromodomain proteins of human BRD proteins and half of all human HAT and involved in the DNA damage response HDAC enzymes are dynamically relocalized, following DNA damage, including to the sites of DNA lesions and BRD proteins are key epigenetic regulators, one-third of repair (Miller et al. 2010; Gong and Miller 2013; Gong which we previously identified as being relocalized to et al. 2015). These studies from our lab and others have es- DNA damage after laser-induced DNA damage (Fig. 1A; tablished the importance of acetylation signaling in the Gong et al. 2015). To further investigate the role of BRD DDR (Gong et al. 2016). For example, a variant of proteins in DSB repair, we performed a siRNA screen of the bromo-and-extra-terminal (BET) BRD protein BRD4 all BRD proteins that are widely expressed in cells using has been shown to insulate DNA damage signaling, and well-established HR and NHEJ cell-based reporter sys- the BRD protein ZMYND8 recruits the NuRD chromatin tems (Fig. 1B; Supplemental Fig. S1B; Pierce et al. 1999; remodeling complex to DNA damage sites where it pro- Bennardo et al. 2008). Knockdown efficiencies of individ- motes repression of transcription and HR repair (Floyd ual BRD genes by siRNAs pools were validated by et al. 2013; Gong et al. 2015). Given the demonstrated RT-qPCR analyses (Supplemental Fig.