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
Gene Expression Omnibus
- The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009) &
- The emerging role of nuclear architecture in DNA repair and genome maintenance. Nature Rev. Mol. Cell Biol. 10, 243–254 (2009) &
- Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434, 907–913 (2005) et al.
- DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434, 864–870 (2005) et al.
- Cell-cycle checkpoints and cancer. Nature 432, 316–323 (2004) &
- Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev. 25, 409–433 (2011) &
- A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006) et al.
- CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006) et al.
- The Brd4 extraterminal domain confers transcription activation independent of pTEFb by recruiting multiple proteins, including NSD3. Mol. Cell. Biol. 31, 2641–2652 (2011) et al.
- Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol. Cell 19, 535–545 (2005) et al.
- The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol. Cell 19, 523–534 (2005) et al.
- Global chromatin compaction limits the strength of the DNA damage response. J. Cell Biol. 178, 1101–1108 (2007) et al.
- Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM- and KAP-1 dependent pathway. Nature Cell Biol. 8, 870–876 (2006) et al.
- γH2AX foci form preferentially in euchromatin after ionising-radiation. PLoS ONE 2, e1057 (2007) et al.
- Heterochromatin is refractory to γ-H2AX modification in yeast and mammals. J. Cell Biol. 178, 209–218 (2007) , , , &
- Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149, 214–231 (2012) et al.
- Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010) et al.
- Chemical phylogenetics of histone deacetylases. Nature Chem. Biol. 6, 238–243 (2010) et al.
- The Smc complexes in DNA damage response. Cell Biosci. 2, 5 (2012) &
- A cooperative activation loop among SWI/SNF, γ-H2AX and H3 acetylation for DNA double-strand break repair. EMBO J. 29, 1434–1445 (2010) , , , &
- Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004)
- Micrococcal nuclease-Southern blot assay: I. MNase and restriction digestions. CSH Protoc. 2007, http://dx.doi.org/10.1101/pdb.prot4890 (2007) &
- Supplementary Figures (1.5 MB)
This file contains Supplementary Figures 1-14.
- 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.
- 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.
- 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.
- 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.