The bromodomain protein Brd4 insulates chromatin from DNA damage signalling

Journal name:
Nature
Volume:
498,
Pages:
246–250
Date published:
DOI:
doi:10.1038/nature12147
Received
Accepted
Published online

DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death1. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response2. 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.

At a glance

Figures

  1. Brd4 isoform B suppresses H2AX phosphorylation after ionizing radiation.
    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 1h after 10Gy ionizing radiation (details of screening assay in Supplementary Figs 1–4). b, γH2AX foci size (upper panel) and mean γH2AX foci per nucleus (lower panel) after 10Gy ionizing radiation (IR) from cells expressing indicated shRNAs (bars show mean and two standard deviations of control values). RFP, red fluorescent protein. 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. aa, amino acid; Iso., isoform. d, H2AX phosphorylation in cells expressing Flag-tagged Brd4 isoform B (arrowheads) or A and C (arrows) at 1h after 10Gy IR. Left: representative images. Middle: quantification of 10 fields from two independent experiments with mean γH2AX signal normalized to untransfected cells. Right: immunoblot of isoform expression levels in whole-cell lysates and anti-Flag immunoprecipitates. Con., control. e, Isoform-specific Brd4 knockdown in cells transfected with the indicated siRNA and analysed by quantitative real-time PCR with reverse transcription (n = 3). f, H2AX phosphorylation levels 1h after indicated ionizing radiation exposure in cells transfected with isoform-specific siRNA (n = 3). Inset shows representative immunoblot for triplicate samples. Data are from U2OS cells. Error bars, s.e.m.; P values were determined using Student’s t-test in this and all subsequent figures unless otherwise indicated.

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

    a, Pulsed-field electrophoresis analysis of DNA from stable cell lines expressing indicated shRNA after 10Gy IR (n = 3). b, Left: micrococcal nuclease assay of control or Brd4 knockdown cells. Right: line traces of representative gel lanes. c, Chromatin structure from cells expressing Flag-tagged Brd4 isoform B (arrowheads) or A and C (arrows) shown by DAPI staining. d, Three-dimensional representation of nuclear DAPI staining intensity from cells in c as indicated by coloured frames. e, DAPI pixel correlation from Brd4 isoform A, B, C and untransfected control cells (n = 3). f, Immunoblots (top) and quantification (bottom) of H2AX phosphorylation after 250nM DMSO, or active (+) and inactive (−) JQ1 at 1h after 10Gy ionizing radiation (n = 3). g, γH2AX signal 1h after 10Gy IR in cells expressing green fluorescent protein (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 250nM (−) JQ1 (inactive) or (+) JQ1 as indicated.

  3. Brd4 isoform B interaction with the condensin complex affects H2AX phosphorylation.
    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 6h after 10Gy 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 relation to DNA damage signalling with protein–protein and kinase–substrate interactions collated from the literature. Protein–protein and kinase–substrate interactions shown by solid and dotted lines, respectively. Colours indicate condensin complex (blue), cohesin complex (pink), other SMC protein complexes (green), cell-cycle regulators (orange) and DNA damage signalling machinery (mint). Diamonds show mass spectrometry and high-content screening hits from a and b. Border colours denote overlap of screens from c. The new interaction of Brd4 with the condensin complex is indicated by the 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 were quantified from ten fields of two independent experiments normalized to control cells. h, H2AX phosphorylation 1h after 10Gy 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 with or without SMC2 knockdown. Data are from ten fields of two independent experiments, as in h, normalized to control untransfected cells.

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

    a, Loss of DNA damage signalling in cells expressing Brd4 isoform B. Left: representative images stained for indicated DDR proteins 1h after 10Gy IR. Arrowheads indicate isoform-B-expressing cells. Right: quantification of ten representative fields from two independent experiments normalized to untransfected cells. b, Cell death 24h after 10Gy IR in cells expressing wild-type or bromodomain-1-mutant isoform B (Isoform B mut. BD1) scored for cleaved caspase 3 by flow cytometry (n = 3). c, Ionizing-radiation-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 several human cancer cell types commonly treated with radiotherapy. No IR, no ionizing radiation. f, Radiation survival effects of JQ1 in glioma cell lines measured at 72h by CellTiterGlo (n = 3). g, Model for Brd4 effects on DNA damage signalling.

Accession codes

Primary accessions

Gene Expression Omnibus

References

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Author information

Affiliations

  1. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Scott R. Floyd,
    • 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,
    • Forest M. White &
    • Michael B. Yaffe
  2. Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA

    • Scott R. Floyd
  3. Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Michael E. Pacold
  4. Whitehead Institute, Cambridge, Massachusetts 02139, USA

    • Michael E. Pacold &
    • David M. Sabatini
  5. Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA

    • David E. Root,
    • Anne E. Carpenter,
    • William C. Hahn,
    • David M. Sabatini,
    • James E. Bradner &
    • Michael B. Yaffe
  6. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • William C. Hahn,
    • Clark C. Chen &
    • James E. Bradner
  7. Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA

    • Clark C. Chen &
    • Michael B. Yaffe
  8. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Forest M. White &
    • Michael B. Yaffe
  9. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    • Michael B. Yaffe

Contributions

S.R.F. and M.B.Y. designed the study, supervised the experiments, analysed the data and wrote the manuscript. D.E.R., W.C.H. and D.M.S. were involved in the design and preparation of the lentiviral shRNA library. S.R.F., M.E.P. and E.B. performed the image-based high-content screen and initial analysis. A.E.C. aided in digital image analysis. S.R.F., Q.H., S.M.C., F.C.L., I.G.C., M.J.L., A.F., R.H., B.A.G., G.C.C. and A.M. performed biochemical, cell biological and molecular biological experiments. B.D.B., A.M.D. and F.M.W. performed mass spectrometry experiments and analysis. J.R. performed bioinformatics analysis. J.E.B. contributed JQ1 compounds and cell lines. S.R.F. and M.B.Y. designed and supervised the experiments. C.C.C., J.E.B. and F.M.W. contributed to the intellectual development of the study and technical writing of the manuscript. All authors contributed to editing the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

The expression profiling Affymetrix u133 plus dataset has been deposited in the NCBI Gene Expression Omnibus database under accession number GSE30700.

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Supplementary information

PDF files

  1. Supplementary Figures (1.5 MB)

    This file contains Supplementary Figures 1-14.

Excel files

  1. Supplementary Table 1 (1.5 MB)

    This file contains γH2AX and nuclear features from high-content shRNA screen. U2OS cells were screened in 384-well plate format using shRNA directed against the indicated gene symbols by the method as outlined in Figure 1 and in the Online Methods. Listed are measurements for several features of identified nuclei and γH2AX foci for each hairpin at all timepoints.

  2. Supplementary Table 2 (44 KB)

    This file contains modulators of γH2AX foci number, intensity and size. List of the top quartile of genes ranked by increasing γH2AX foci number per nucleus, area, and integrated fluorescence intensity 1 and 6 hours following 10 Gy IR. Genes that appear shaded in green scored in the top quartile at both the 1 and 6 hour time points.

  3. Supplementary Table 3 (33 KB)

    This file contains Brd4 isoform B interacting proteins. A list of peptides, associated genes, and MASCOT scores identified as Brd4 isoform B interactors by mass spectrometry from U2OS cell immunoprecipitates from two independent experiments. Genes that appear shaded in green were identified in experimental replicates 1 and 2.

  4. Supplementary Table 4 (41 KB)

    This file contains Brd4 isoform interactions with SMC proteins. A list of protein scores, unique and total number of peptides, peptide sequences and MASCOT scores for peptides identified by mass spectrometry of Commassie Brilliant Blue stained gel regions as indicated in Supplementary Figure 7.

Additional data