The bromodomain protein Brd4 insulates chromatin from DNA damage signalling - PubMed (original) (raw)

. 2013 Jun 13;498(7453):246-50.

doi: 10.1038/nature12147. Epub 2013 Jun 2.

Michael E Pacold, Qiuying Huang, Scott M Clarke, Fred C Lam, Ian G Cannell, Bryan D Bryson, Jonathan Rameseder, Michael J Lee, Emily J Blake, Anna Fydrych, Richard Ho, Benjamin A Greenberger, Grace C Chen, Amanda Maffa, Amanda M Del Rosario, David E Root, Anne E Carpenter, William C Hahn, David M Sabatini, Clark C Chen, Forest M White, James E Bradner, Michael B Yaffe

Affiliations

The bromodomain protein Brd4 insulates chromatin from DNA damage signalling

Scott R Floyd et al. Nature. 2013.

Abstract

DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Brd4 isoform B suppresses H2AX phosphorylation after ionizing radiation

a, Rank of hairpins from shRNA screen ordered by integrated γH2AX foci intensity at 1 hr following 10 Gy IR (details of screening assay in Supplementary Figs. 1–4). b,γH2AX foci size (upper panel), and mean γH2AX foci per nucleus (lower panel) after 10 Gy IR from cells expressing indicated shRNAs (bars show mean and 2 S.D. of control values). c, Domain structure of Brd4 isoforms showing conserved tandem bromodomains (BRD), extra-terminal (ET) domain, siRNA and antibody target sequences, and unique isoform B exon. d, H2AX phosphorylation in cells expressing FLAG-tagged Brd4 isoform B (arrowheads) or A and C (arrows) at 1 hr after 10 Gy IR. Left: representative images. Middle: quantification of 10 fields from 2 independent experiments with mean γH2AX signal normalized to untransfected cells. Right: Immunoblot of isoform expression levels in whole cell lysates and anti-FLAG immunoprecipitates. e, Isoform-specific Brd4 knockdown in cells transfected with the indicated siRNA and analysed by quantitative real-time RT-PCR (n=3). f, H2AX phosphorylation levels 1 hr after indicated IR exposure in cells transfected with isoform-specific siRNA (n=3). Inset shows representative immunoblot for triplicate samples. Data are from U2OS cells. Error bars indicate S.E.M. and p-values were determined using Student’s t-test in this and all subsequent figures unless otherwise indicated.

Figure 2

Figure 2. Brd4 isoform B limits H2AX phosphorylation via bromodomain-acetyl lysine mediated effects on chromatin structure

a, Pulsed-field electrophoresis analysis of DNA from stable cell lines expressing indicated shRNA after 10 Gy IR (n=3). b, Left: Micrococcal nuclease assay of control or Brd4 knockdown cells. Right: Line traces of representative gel lanes as in left panel. c, Chromatin structure from cells expressing FLAG-tagged Brd4 isoform B (arrowheads) or A and C (arrows) revealed by DAPI staining. d, 3D representation of nuclear DAPI staining intensity from cells in (c) as indicated by colored frames. e, DAPI pixel correlation from Brd4 isoform A, B, C and untransfected control cells (n=3). f, Immunoblots (upper panels) and quantification (lower panels) of H2AX phosphorylation following 250 nM DMSO, or active (+) and inactive (−) JQ1 at 1 hr after 10 Gy IR (n=3). g, γH2AX signal 1 hr after 10 Gy IR in cells expressing GFP-wild-type Brd4 isoform B (arrowheads), isoform B with mutations that abrogate acetyl lysine binding of bromodomain 1 (BD1) or 2 (BD2) (arrows), or wild-type Brd4 isoform B in the presence of 250 nM (−) JQ1 (inactive) or (+) JQ1 as indicated.

Figure 3

Figure 3. Brd4 isoform B interaction with the condensin complex affects H2AX phosphorylation

a, Mass spectrometry identification of co-immunoprecipitated proteins from FLAG-tagged Brd4 isoform B-expressing cells. b, Identification of candidate Brd4 interactors by ranking chromatin modifier shRNAs from screen for elevated H2AX foci intensity, area and number at 1 and 6 hr after 10 Gy IR. Dashed red lines indicate top quartile. c, Intersection of two independent mass-spectrometry experiments (a) with the top quartile of candidates in (b). Overlapping set includes Brd4, SMC2 and NCAPD3. d, Network representation of SMC proteins and relationship to DNA damage signaling with protein-protein and kinase-substrate interactions collated from the literature. Protein-protein and kinase-substrate interactions shown by solid and dotted lines, respectively. Colors indicate condensin complex (blue), cohesin complex (pink), other SMC protein complexes (green), cell cycle regulators (orange) and DNA damage signaling machinery (mint). Diamonds show mass spectrometry and HCS hits from (a-b). Border colors denote overlap of screens from (c). The novel interaction of Brd4 with the condensin complex is indicated by red line. e, Validation of isoform B-condensin interaction with blotting immunoprecipitates from cells transfected with indicated FLAG-tagged constructs. f, Immunoblot verification of SMC2 knockdown from cells transfected with SMC2 siRNA. g, Nuclear γH2AX signal from cells transfected with indicated combinations of control DNA, Brd4 isoform B, and/or SMC2 siRNA. Data was quantified from 10 fields of 2 independent experiments normalized to control cells. h, H2AX phosphorylation 1 hr after 10 Gy IR in cells simultaneously expressing isoform B and control (arrows) or SMC2 siRNA (arrowheads). i, Chromatin staining pattern in cells simultaneously expressing isoform B and control (red frame) or SMC2 (blue frame) siRNA. j, Mean nuclear γH2AX signal in GFP-isoform B expressing cells +/− SMC2 knock-down. Data is from 10 fields of 2 independent experiments as in (h) normalized to control untransfected cells.

Figure 4

Figure 4. Brd4 isoform B affects ionizing radiation-induced cell cycle checkpoints and survival

a, Loss of DNA damage signaling in cells expressing Brd4 isoform B. Left: representative images stained for indicated DDR proteins 1 hr after 10 Gy IR. Arrowheads indicate isoform B-expressing cells. Right: quantitation of 10 representative fields from 2 independent experiments normalized to untransfected cells. b, Cell death 24 hr after 10 Gy IR in cells expressing WT or bromodomain 1-mutant isoform B scored for cleaved caspase 3 by flow cytometry (n=3). c, IR-induced cell cycle arrest and recovery in Brd4 isoform knockdown cells assayed by propidium iodide staining and flow cytometry. d, Cell survival after irradiation in Brd4 isoform knockdown cells measured by colony formation. e, JQ1 effect on γH2AX in multiple human cancer cell types commonly treated with radiotherapy. f, Radiation survival effects of JQ1 in glioma cell lines measured at 72 hr by CellTiterGlo (n=3). g, Model for Brd4 effects on DNA damage signaling.

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