The bromodomain and extra-terminal motif (BET) protein BRD4 binds to acetylated histones at enhancers and promoters via its bromodomains (BDs) to regulate transcriptional elongation. In human colorectal cancer cells, we found that BRD4 was recruited to enhancers that were co-occupied by mutant p53 and supported the synthesis of enhancer-directed transcripts (eRNAs) in response to chronic immune signaling. BRD4 selectively associated with eRNAs that were produced from BRD4-bound enhancers. Using biochemical and biophysical methods, we found that BRD4 BDs function cooperatively as docking sites for eRNAs and that the BDs of BRD2, BRD3, BRDT, BRG1, and BRD7 directly interact with eRNAs. BRD4-eRNA interactions increased BRD4 binding to acetylated histones in vitro and augmented BRD4 enhancer recruitment and transcriptional cofactor activities. Our results suggest a mechanism by which eRNAs are directly involved in gene regulation by modulating enhancer interactions and transcriptional functions of BRD4.
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Calo, E. & Wysocka, J. Modification of enhancer chromatin: what, how, and why? Mol. Cell 49, 825–837 (2013).
Heinz, S., Romanoski, C. E., Benner, C. & Glass, C. K. The selection and function of cell-type-specific enhancers. Nat. Rev. Mol. Cell Biol. 16, 144–154 (2015).
Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).
Thurman, R. E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).
Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).
Chepelev, I., Wei, G., Wangsa, D., Tang, Q. & Zhao, K. Characterization of genome-wide enhancer-promoter interactions reveals coexpression of interacting genes and modes of higher order chromatin organization. Cell Res. 22, 490–503 (2012).
Creyghton, M. P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).
Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).
Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).
Filippakopoulos, P. & Knapp, S. The bromodomain interaction module. FEBS Lett. 586, 2692–2704 (2012).
Marushige, K. Activation of chromatin by acetylation of histone side chains. Proc. Natl. Acad. Sci. USA 73, 3937–3941 (1976).
Filippakopoulos, P. et al. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149, 214–231 (2012).
Umehara, T. et al. Structural basis for acetylated histone H4 recognition by the human BRD2 bromodomain. J. Biol. Chem. 285, 7610–7618 (2010).
Morinière, J. et al. Cooperative binding of two acetylation marks on a histone tail by a single bromodomain. Nature 461, 664–668 (2009).
Dhalluin, C. et al. Structure and ligand of a histone acetyltransferase bromodomain. Nature 399, 491–496 (1999).
Mujtaba, S., Zeng, L. & Zhou, M. M. Structure and acetyl-lysine recognition of the bromodomain. Oncogene 26, 5521–5527 (2007).
Yang, X. J. Lysine acetylation and the bromodomain: a new partnership for signaling. BioEssays 26, 1076–1087 (2004).
Zeng, L. & Zhou, M. M. Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 513, 124–128 (2002).
Dawson, M. A. et al. Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 478, 529–533 (2011).
Delmore, J. E. et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904–917 (2011).
Zuber, J. et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478, 524–528 (2011).
Huang, B., Yang, X. D., Zhou, M. M., Ozato, K. & Chen, L. F. Brd4 coactivates transcriptional activation of NF-κB via specific binding to acetylated RelA. Mol. Cell. Biol. 29, 1375–1387 (2009).
Zou, Z. et al. Brd4 maintains constitutively active NF-κB in cancer cells by binding to acetylated RelA. Oncogene 33, 2395–2404 (2014).
Wu, S. Y., Lee, A. Y., Lai, H. T., Zhang, H. & Chiang, C. M. Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting. Mol. Cell 49, 843–857 (2013).
Brown, J. D. et al. NF-κB directs dynamic super enhancer formation in inflammation and atherogenesis. Mol. Cell 56, 219–231 (2014).
Roe, J. S., Mercan, F., Rivera, K., Pappin, D. J. & Vakoc, C. R. BET bromodomain inhibition suppresses the function of hematopoietic transcription factors in acute myeloid leukemia. Mol. Cell 58, 1028–1039 (2015).
Shi, J. et al. Disrupting the interaction of BRD4 with diacetylated Twist suppresses tumorigenesis in basal-like breast cancer. Cancer Cell 25, 210–225 (2014).
Stewart, H. J., Horne, G. A., Bastow, S. & Chevassut, T. J. BRD4 associates with p53 in DNMT3A-mutated leukemia cells and is implicated in apoptosis by the bromodomain inhibitor JQ1. Cancer Med. 2, 826–835 (2013).
Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).
Chen, J. et al. BET inhibition attenuates Helicobacter pylori–induced inflammatory response by suppressing inflammatory gene transcription and enhancer activation. J. Immunol. 196, 4132–4142 (2016).
Hah, N. et al. Inflammation-sensitive super enhancers form domains of coordinately regulated enhancer RNAs. Proc. Natl. Acad. Sci. USA 112, E297–E302 (2015).
Jang, M. K. 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).
Winter, G. E. et al. BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol. Cell 67, 5–18 (2017).
Kanno, T. et al. BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nat. Struct. Mol. Biol. 21, 1047–1057 (2014).
Nagarajan, S. et al. Bromodomain protein BRD4 is required for estrogen receptor–dependent enhancer activation and gene transcription. Cell Rep. 8, 460–469 (2014).
Andersson, R. et al. An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461 (2014).
Arner, E. et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010–1014 (2015).
Hsieh, C. L. et al. Enhancer RNAs participate in androgen receptor–driven looping that selectively enhances gene activation. Proc. Natl. Acad. Sci. USA 111, 7319–7324 (2014).
Lai, F., Gardini, A., Zhang, A. & Shiekhattar, R. Integrator mediates the biogenesis of enhancer RNAs. Nature 525, 399–403 (2015).
Lam, M. T. et al. Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription. Nature 498, 511–515 (2013).
Li, W. et al. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498, 516–520 (2013).
Melo, C. A. et al. eRNAs are required for p53-dependent enhancer activity and gene transcription. Mol. Cell 49, 524–535 (2013).
Mousavi, K. et al. eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci. Mol. Cell 51, 606–617 (2013).
Rahnamoun, H. et al. Mutant p53 shapes the enhancer landscape of cancer cells in response to chronic immune signaling. Nat. Commun. 8, 754 (2017).
Schaukowitch, K. et al. Enhancer RNA facilitates NELF release from immediate early genes. Mol. Cell 56, 29–42 (2014).
Lai, F. et al. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494, 497–501 (2013).
Bose, D. A. et al. RNA binding to CBP stimulates histone acetylation and transcription. Cell 168, 135–149 (2017).
Wu, S. Y. & Chiang, C. M. The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J. Biol. Chem. 282, 13141–13145 (2007).
Mertz, J. A. et al. Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proc. Natl. Acad. Sci. USA 108, 16669–16674 (2011).
Ott, C. J. et al. BET bromodomain inhibition targets both c-Myc and IL7R in high-risk acute lymphoblastic leukemia. Blood 120, 2843–2852 (2012).
Yap, K. L. et al. Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by Polycomb CBX7 in transcriptional silencing of INK4a. Mol. Cell 38, 662–674 (2010).
Devaiah, B. N. et al. BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin. Nat. Struct. Mol. Biol. 23, 540–548 (2016).
Fujisawa, T. & Filippakopoulos, P. Functions of bromodomain-containing proteins and their roles in homeostasis and cancer. Nat. Rev. Mol. Cell Biol. 18, 246–262 (2017).
Sanchez, R., Meslamani, J. & Zhou, M. M. The bromodomain: from epigenome reader to druggable target. Biochim. Biophys. Acta 1839, 676–685 (2014).
Miller, T. C. et al. A bromodomain-DNA interaction facilitates acetylation-dependent bivalent nucleosome recognition by the BET protein BRDT. Nat. Commun. 7, 13855 (2016).
Morrison, E. A. et al. DNA binding drives the association of BRG1/hBRM bromodomains with nucleosomes. Nat. Commun. 8, 16080 (2017).
Ruthenburg, A. J., Li, H., Patel, D. J. & Allis, C. D. Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8, 983–994 (2007).
Dey, A., Chitsaz, F., Abbasi, A., Misteli, T. & Ozato, K. The double bromodomain protein Brd4 binds to acetylated chromatin during interphase and mitosis. Proc. Natl. Acad. Sci. USA 100, 8758–8763 (2003).
Lauberth, S. M. et al. H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation. Cell 152, 1021–1036 (2013).
Lauberth, S. M., Bilyeu, A. C., Firulli, B. A., Kroll, K. L. & Rauchman, M. A phosphomimetic mutation in the Sall1 repression motif disrupts recruitment of the nucleosome remodeling and deacetylase complex and repression of Gbx2. J. Biol. Chem. 282, 34858–34868 (2007).
We are grateful to C.-M. Chiang (University of Texas Southwestern) for providing the pF:hBRD4 (1–722)-11d, pcDNA3-F:hBRD4 FL, and pcDNA3-F:hBRD4 ∆BD1/2 plasmids and the BRD4-FL- and BRD4 ∆BD1/2–expressing baculovirus. We are also thankful to X. Chen (University of California, Davis) for providing the SW480 shLacZ and shp53 cell lines. H.R. was supported by the University of California at San Diego Cellular and Molecular Genetics Training Program through an institutional grant from the National Institute of General Medicine (T32 GM007240). This work was supported by Research Scholar Award from the Sidney Kimmel Foundation for Cancer Research 857A6A (S.M.L.), American Cancer Society ACS-IRG 70-002 (S.M.L.), and the University of California Cancer Research Coordinating Committee, CRN-17-420616 (S.M.L.).
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 BRD4 cooperates with mutp53 at active enhancers upon chronic immune signaling.
a, Venn diagram depicting the overlap between p53R273H,P309S and BRD4 binding at H3K27ac- and H3K4me1-enriched, intergenic regions in SW480 cells after 16 h of TNF-α treatment. b, De novo motif analyses of BRD4 and p53R273H, P309S co-bound ChIP-seq peaks at active enhancers. c, Sequential ChIP–qPCR with p53 immunoprecipitation followed by BRD4 or IgG immunoprecipitation in SW480 cells after 16 h of TNF-α treatment, at enhancer (A) and nonspecific (B) regions of MMP9 and CCL2. d, Immunoblot analysis of BRD4 coimmunoprecipitation with lysates from SW480 cells treated with TNF-α for 0 or 16 h. e, Immunoblot analysis of BRD4 and p53 (wild type and R273H) interactions (top) using recombinant proteins analyzed by Coomassie staining (bottom). n = 3 independent coimmunoprecipitation and binding assays. f, Immunoblot analysis of SW480 cells expressing LacZ (Ctrl) or p53 shRNA and treated with TNF-α for 0 or 16 h. g, qRT–PCR analysis of MMP9 and CCL2 eRNAs and mRNAs in SW480 cells expressing control (Ctrl) or BRD4 shRNA (BRD4-2) and treated with TNF-α for 0 or 16 h. h, ChIP–qPCR of IgG and p53R273H,P309S enrichment at enhancer (A) and nonspecific (B) regions of MMP9 and CCL2 in SW480 cells expressing Ctrl or BRD4 shRNA and treated with TNF-α for 0 or 16 h. i, Immunoblot and qRT–PCR analysis of SW480 cells treated with vehicle or JQ1 and with TNF-α for 0 or 16 h. In g and i, expression levels following TNF-α treatment are relative to the levels before treatment and data represent the mean and s.e.m. of n= 3 independent experiments. In c and h, data represent the mean and s.e.m. of n = 2 independent experiments that are representative of three replicates. Statistical significance was determined by two-tailed Student’s t test. *P < 0.05
a, qRT–PCR and immunoblot analyses of SW480 cells treated with TNF-α for 0 or 16 h. b, qRT–PCR analysis of the CSF2 and TNFAIP3 eRNAs following UV-RIP using IgG or BRD4 antibodies in SW480 cells treated with TNF-α for 0 or 16 h. Enrichment levels for each TNF-α-treated immunoprecipitation are relative to the levels before TNF-α treatment. In a and b, data represent the mean and s.e.m. of n = 3 independent experiments. Statistical significance was determined by two-tailed Student’s t test. *P < 0.05. c, UCSC Genome Browser images of GRO-seq and BRD4 ChIP-seq signals in SW480 cells treated as described in a at active enhancer regions of CSF2 and TNFAIP3. d, SYBR Gold staining of an in vitro binding assay between BRD4-FL and various in vitro–transcribed RNA molecules. n = 3 independent experiments. e, Quantification of RNA EMSAs to determine the fraction of RNA bound to BRD4-FL as shown in Fig. 3a and BRD4 ΔBD1/2 as shown in Fig. 3e
Supplementary Figure 3 eRNAs enhance BRD4 binding to acetylated histone peptides and octamers in vitro.
a, Recombinant FLAG-p300 protein analyzed by Coomassie staining. b, Immobilized peptide pulldown assay using biotinylated H4 peptides (unmodified or K16 acetylated) with either recombinant BRD4-FL or BRD4 ΔBD1/2 in the absence or presence of refolded CCL2 eRNA as indicated. Recombinant BRD4-FL and BRD4 ΔBD1/2 were detected by immunoblotting with an antibody specific to BRD4. c, Immunoblot analysis of in vitro binding assays with unacetylated or acetylated histone octamers, recombinant BRD4-FL or ΔBD1/2, and refolded CCL2 eRNA as indicated. d, In vitro histone octamer binding assay as described in c with recombinant BRD4-FL and increasing doses of refolded MMP9 eRNA (0.06, 0.1, 0.2, 1, and 2 nM). In c and d, immunoblot analysis with H3K27ac and H3K9ac antibodies confirmed p300/acetyl-CoA-mediated acetylation of histone octamers. Lower panels in b–d represent the loading of the indicated proteins by Coomassie staining. n = 3 independent experiments for all in vitro binding assays
Supplementary Figure 4 eRNA depletion reduces the expression of corresponding mRNAs and impacts BRD4 binding.
a, qRT–PCR analysis of MMP9 and CCL2 eRNAs and mRNAs in SW480 cells expressing control or a second shRNA oligonucleotide against MMP9 and CCL2 eRNAs and treated with TNF-α for 0 or 16 h. b, qRT–PCR analysis of CPA4 and CYP24A1 eRNAs and mRNAs in SW480 cells treated as described in Fig. 5a. c,d, Immunoblot (c) and qRT–PCR analysis (d) of SW480 cells treated with TNF-α for 0 or 16 h and treated with Act D for 2 h. In a, b, and d, the expression levels following TNF-α treatment are relative to the levels before TNF-α exposure and data represent the mean and s.e.m. of n = 3 independent experiments. e, ChIP–qPCR analyses of IgG and BRD4 enrichment in SW480 cells treated as described in d, at the enhancer (A) and nonspecific (B) regions of the MMP9 and CCL2 gene loci. Data represent the mean and s.e.m. of n = 2 independent ChIP experiments that are representative of at least three replicates. Statistical significance was determined by two-tailed Student’s t test. *P < 0.05
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Rahnamoun, H., Lee, J., Sun, Z. et al. RNAs interact with BRD4 to promote enhanced chromatin engagement and transcription activation. Nat Struct Mol Biol 25, 687–697 (2018). https://doi.org/10.1038/s41594-018-0102-0
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