Brd4 Binds to Active Enhancers to Control Cell Identity Gene Induction in Adipogenesis and Myogenesis Ji-Eun Lee

Home , DBP (gene), MLL4, NFIC (gene)

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2017 Brd4 binds to active enhancers to control cell identity gene induction in adipogenesis and myogenesis Ji-Eun Lee

Young-Kwon Park

Sarah Park

Younghoon Jang

Nicholas Waring George Washington University

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APA Citation Lee, J., Park, Y., Park, S., Jang, Y., Waring, N., Dey, A., Ozato, K., Lai, B., Peng, W., & Ge, K. (2017). Brd4 binds to active enhancers to control cell identity gene induction in adipogenesis and myogenesis. Nature Communications, (). http://dx.doi.org/10.1038/ s41467-017-02403-5

This Journal Article is brought to you for free and open access by the Anatomy and Regenerative Biology at Health Sciences Research Commons. It has been accepted for inclusion in Anatomy and Regenerative Biology Faculty Publications by an authorized administrator of Health Sciences Research Commons. For more information, please contact [email protected]. Authors Ji-Eun Lee, Young-Kwon Park, Sarah Park, Younghoon Jang, Nicholas Waring, Anup Dey, Keiko Ozato, Binbin Lai, Weiqun Peng, and Kai Ge

This journal article is available at Health Sciences Research Commons: https://hsrc.himmelfarb.gwu.edu/smhs_anatregbio_facpubs/ 163 ARTICLE

DOI: 10.1038/s41467-017-02403-5 OPEN Brd4 binds to active enhancers to control cell identity gene induction in adipogenesis and myogenesis

Ji-Eun Lee 1, Young-Kwon Park1, Sarah Park1, Younghoon Jang1, Nicholas Waring1,2, Anup Dey3, Keiko Ozato3, Binbin Lai1,2, Weiqun Peng2 & Kai Ge 1

1234567890 The epigenomic reader Brd4 is an important drug target for cancers. However, its role in cell differentiation and animal development remains largely unclear. Using two conditional knockout mouse strains and derived cells, we demonstrate that Brd4 controls cell identity gene induction and is essential for adipogenesis and myogenesis. Brd4 co-localizes with lineage-determining transcription factors (LDTFs) on active enhancers during differentiation. LDTFs coordinate with H3K4 mono-methyltransferases MLL3/MLL4 (KMT2C/KMT2D) and H3K27 acetyltransferases CBP/p300 to recruit Brd4 to enhancers activated during differentiation. Brd4 deletion prevents the enrichment of Mediator and RNA polymerase II transcription machinery, but not that of LDTFs, MLL3/MLL4-mediated H3K4me1, and CBP/ p300-mediated H3K27ac, on enhancers. Consequently, Brd4 deletion prevents enhancer RNA production, cell identity gene induction and cell differentiation. Interestingly, Brd4 is dispensable for maintaining cell identity genes in differentiated cells. These ﬁndings identify Brd4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation.

1 Adipocyte Biology and Gene Regulation Section, Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. 2 Departments of Physics and Anatomy and Regenerative Biology, The George Washington University, Washington, DC 20052, USA. 3 Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. Correspondence and requests for materials should be addressed to K.G. (email: [email protected])

NATURE COMMUNICATIONS | 8: 2217 | DOI: 10.1038/s41467-017-02403-5 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02403-5

ifferentiation is controlled by cell-type-specific gene Adipogenesis and myogenesis are two model systems that are expression, which is under the control of transcriptional well suited for studying cell differentiation. In adipogenesis, the D 1 enhancers . Enhancers possess recognition motifs for induction of early adipogenic transcription factors (TFs), sequence-specific lineage-determining transcription factors including CCAAT/enhancer binding protein-β (C/EBPβ), in turn (LDTFs), which bind to and activate enhancers2. LDTFs recruit induces the expression of two master adipogenic TFs, peroxisome epigenomic regulators that remodel the chromatin landscape by proliferator-activated receptor-γ (PPARγ) and C/EBPα15,16. adding epigenetic modifications (i.e., methylation, acetylation, PPARγ and C/EBPα work in cooperation to activate many etc.) to the histone tails of the associated nucleosomes, after adipocyte-specific genes. The synchronized nature of adipogen- which RNA polymerase II (Pol II) is recruited and transcription esis in cell culture, wherein the majority of the cells in the con- of enhancer RNA (eRNA) and nearby genes occurs2. Enhancers fluent population differentiate from preadipocytes to mature are enriched with histone H3K4 mono-methylation (H3K4me1), adipocytes within 6–8 days, allows for a system conducive to which is mainly deposited by H3K4 methyltransferases MLL3 studying gene expression during the process of differentiation17. (KMT2C) and MLL4 (KMT2D)3. H3K4me1 precedes the addi- Myogenesis is another model of synchronized cell differentiation. tion of the active enhancer mark H3K27ac by histone acetyl- Myogenic differentiation protein (MyoD) and myogenic factor 5 transferases CBP/p3004,5. CBP/p300 binding identifies active (Myf5) are required for commitment to the muscle differentiation enhancers that control cell-type-specific gene expression6. The program, while myogenin plays a necessary role in establishing acetyl marks on histones act as docking sites for epigenomic the terminal muscle phenotype18. readers that have conserved bromodomains7. Brd4 is a prominent drug target for cancers but its role in The epigenomic reader Brd4 is a member of the bromodomain normal cell differentiation and tissue development is largely and extra-terminal domain (BET) family of nuclear proteins that unexplored. In this study, we use adipogenesis and myogenesis as also include Brd2 and Brd38,9. Brd4 is enriched on active model systems to explore the role of Brd4 in differentiation and enhancers and promoters10,11. Cooperation between LDTFs and development. Using tissue-specific Brd4 KO mice, we provide CBP/p300 facilitates Brd4 recruitment to its target promoters and in vivo evidence that Brd4 is essential for adipogenesis and enhancers11. Small-molecule competitive BET inhibitors such as myogenesis. During adipogenesis, Brd4 preferentially binds to JQ1 bind to the acetyl-lysine binding pocket of the BET bro- active enhancers and controls the induction of cell-type-specific modomains and displaces BET proteins from chromatin12. JQ1- genes. Furthermore, we determine the sequential actions of mediated inhibition of Brd4 binding leads to the displacement of LDTFs, enhancer epigenomic writers MLL3/MLL4 and CBP/ the Mediator complex and Pol II from enhancers, which in turn p300, Brd4, transcription coactivator complex Mediator and reduces eRNA production and associated gene expression10,13,14. general transcription factor TFIID, Pol II, and transcription

a Brd4f/f 34567 WT allele

Flox allele

KO allele

LoxP

bdf/f f/+ Brd4 × Brd4 ; Myf5-Cre Brd4f/f Brd4f/f ; Myf5-Cre Genotype Number of Survival (E18.5) embryos after isolation M Brd4f/+ 17 17 BAT Brd4f/+ ; Myf5-Cre 15 15 BAT Brd4f/f 17 17 Brd4f/f ; Myf5-Cre 14 0 BAT c Brd4f/f Brd4f/f ; Myf5-Cre

UCP1 Myosin

Fig. 1 Brd4 is required for the development of brown adipose tissue and muscle. Brd4f/f mice were crossed with Myf5-Cre mice. a Schematics of wild-type (WT) allele, conditional KO (flox) allele, and KO allele of Brd4f/f mice. In the flox allele, exon 3 was flanked by two loxP sites. b Genotype of progeny from crossing between Brd4f/+;Myf5-Cre and Brd4f/f mice at E18.5. Brd4f/f;Myf5-Cre mice survived at E18.5 but died within minutes after cesarean section due to muscle defects around rib cage. Similar phenotypes were observed in 14 Brd4f/f;Myf5-Cre mice. c Representative morphology of E18.5 embryos. d Histological analysis of E18.5 embryos. Sagittal sections of cervical/thoracic area were stained with H&E (upper panels) or with antibodies against the brown adipose tissue (BAT) marker UCP1 (green) and the muscle (M) marker myosin (red) (lower panels). Scale bars = 80 μm

2 NATURE COMMUNICATIONS | 8: 2217 | DOI: 10.1038/s41467-017-02403-5 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02403-5 ARTICLE elongation factor p-TEFb in enhancer activation and cell identity Cre mice survived until birth. E18.5 Brd4f/f;Myf5-Cre embryos gene induction during differentiation. We further show that were obtained at the expected Mendelian ratio but were unable to unlike its critical role in cell differentiation, Brd4 is largely dis- breathe and died immediately (Fig. 1b). They showed an abnor- pensable for the maintenance of cell identity gene expression in mal hunched posture due to severe reduction of back muscles differentiated cells. (Fig. 1c). Immunohistochemical analysis of cervical regions of E18.5 embryos revealed that the deletion of Brd4 leads to severe Results reduction of BAT and muscle mass, indicating that Brd4 is Brd4 is required for adipose tissue and muscle development. essential for BAT and muscle development (Fig. 1d). Two independently developed Brd4 conditional knockout (KO) mouse strains, Brd4f/f and Brd4f/f #2, were used in this study. In Brd4 controls cell identity gene induction. To investigate how the Brd4f/f strain, the exon 3 was flanked by two loxP sites Brd4 regulates adipose tissue development, we isolated primary (Fig. 1a), while in the Brd4f/f #2 strain, the exon 5 was floxed Brd4f/f preadipocytes from BAT. After immortalization, cells were (Supplementary Fig 1a). To study the role of Brd4 in adipose infected with adenoviruses expressing green fluorescent protein tissue and muscle development, we crossed Brd4f/f with Myf5-Cre (GFP) or Cre (Fig. 2a, b and Supplementary Fig 2). Deletion of mice to delete Brd4 gene in progenitor cells of brown adipose Brd4 by Cre did not affect the growth rate of SV40T- tissue (BAT) and muscle lineages19,20. The resulting Brd4f/f;Myf5- immortalized brown preadipocytes (Fig. 2c), but prevented adipogenesis and the induction of adipocyte marker genes such as Pparg, Cebpa, and Fabp4 (Fig. 2d, e). We confirmed the essential role of Brd4 in adipogenesis in an independent brown pre- f/f → Brd4 brown preadipocytes adipocytes adipocyte cell line derived from Brd4f/f #2 mice (Supplementary ab c Fig 1b–d). Consistent with the phenotypes observed in both Brd4 120 6 GFP knockout cell lines, knockdown of Brd4 in 3T3-L1 white pre- 100 GFP Cre 5 Cre 80 250 6 4 adipocytes impaired adipogenesis (Supplementary Fig 3). GFP Brd4 60 3 Knockdown of Brd4 in C2C12 myoblast inhibited myogenesis Cre 75 ×10 40 RbBP5 2 – 20 and myocyte gene expression (Supplementary Fig 4a d). Cell number, 1 fi mRNA level (%) 0 0 To nd out how Brd4 regulates gene expression during Brd4 Day 0 1 2 3 4 5 6 adipogenesis, we performed RNA-Seq analyses before (day 0, D0) and during (D2) adipogenesis of Brd4f/f preadipocytes infected deGFP Cre 1.5 120 with adenoviral GFP or Cre. Using a 2.5-fold cutoff for Brd4 Cebpa differential expression, we defined up-regulated (816/16,323), GFP Cre 1.0 80 down-regulated (1045/16,323), and unchanged (14,462/16,323) 0.5 40 genes from D0 to D2 of differentiation (Fig. 2f). Among the 816 up-regulated genes, 351 were induced in a Brd4-dependent 0 0 60 120 manner. Interestingly, only Brd4-dependent up-regulated genes Pparg Fabp4 were strongly associated functionally with fat cell differentiation 40 80 and lipid metabolism (Fig. 2g). During C2C12 myogenesis, Brd4- mRNA level (fold) 20 40 dependent up-regulated genes were preferentially associated functionally with muscle development and function (Supplemen- 0 0 D0 D2 D4 D6 D0 D2 D4 D6 tary Fig 4e, f). These results suggest that Brd4 controls cell identity gene induction during adipogenesis and myogenesis. f RNA-Seq: adipogenesis Brd4 binds to cell identity genes. Next, we performed ChIP-Seq Down-regulated at 14462 Brd4-dependent to map the genomic binding of Brd4 before (D0), during (D2), D2 of adipogenesis and after (D7) adipogenesis. To exclude false-positive genomic Mild or 1045 816 Up-regulated at 351 no change D2 of adipogenesis 465 Fig. 2 Brd4 controls cell identity gene induction during adipogenesis. a–e Brd4-independent Brd4 is required for adipogenesis of brown preadipocytes. SV40T- f/f g immortalized Brd4 brown preadipocytes were infected with adenoviral GO: adipogenesis GFP or Cre, followed by adipogenesis assay. a qRT-PCR confirmation of Gene group GO term P value Brd4 deletion in preadipocytes. b Deletion of Brd4 was confirmed by Brown fat cell differentiation 7.8E–11 western blot analysis. RbBP5 was used as a loading control. c Deletion of Fat cell differentiation 3.6E–10 Brd4 has little effect on the growth of immortalized preadipocytes. d Oil red Triglyceride metabolic process 6.7E–06 O staining at D6 of adipogenesis. Upper panels, stained dishes; lower Up-regulated Oxidation reduction 8.1E–06 panels, representative fields under microscope. Scale bars = 15 μm. e qRT- (Brd4-dependent) Lipid metabolic process 1.3E–05 Acylglycerol metabolic process 2.0E–05 PCR of Brd4, Pparg, Cebpa, and Fabp4 expression at indicated time points Glycerol ether metabolic process 2.6E–05 during adipogenesis. D0/2/4/6, day 0/2/4/6. The data are presented as Neutral lipid metabolic process 2.6E–05 means ± SD. Three technical replicates from a single experiment were Fatty acid metabolic process 3.2E–05 used. f, g Brd4 is required for induction of cell identity genes during Up-regulated Regulation of development 1.8E–05 adipogenesis. Cells were collected before (D0) and during (D2) Cellular response to hormone 2.2E–05 (Brd4-independent) Intracellular signaling cascade 3.2E–05 adipogenesis for RNA-Seq. (f) Schematic of identification of Brd4- Cell adhesion 2.3E–07 dependent and -independent up-regulated genes during adipogenesis. The Down-regulated Regulation of transferase activity 8.4E–05 cutoff for up- or down-regulation is 2.5-fold. The cutoff for Brd4 Regulation of kinase activity 1.2E–04 dependency is 2.5-fold. g Gene ontology (GO) analysis of gene groups Intracellular signaling cascade 2.4E–04 defined in f

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a ChIP-Seq: Brd4 in adipogenesis b e D2 (13676) GO: Brd4-associated genes #1 #2 4145 D0 D2 D7 D0 D2 D7 Cell stages GO term P value 250 Brd4 5438 Apoptotic signaling pathway 6E–121 75 25916 RbBP5 Extracellular structure organization 3E–114

7036 10078 D0 Negative regulation of transferase activity 2E–102 (pre Regulation of kinase activity 2E–99 adipocyte) D0 D7 Collagen fibril organization 4E–99 (37111) (27351) Cell-substrate adhesion 8E–84 mRNA catabolic process 1E–82 c D0 D2 D7 d ChIP-Seq: Brd4 Brown fat cell differentiation 7E–135 Response to peptide hormone stimulus 7E–113 Fat cell differentiation 1E–104 21657 8868 D2 Response to insulin stimulus 2E–103 5694 Insulin receptor signaling pathways 2E–83 Regulation of lipid metabolic process 1E–82

Brd4 binding sites Lipid homeostasis 4E–69 D7 (adipocyte) C2C12 myocyte (27351) (14562) Brown fat cell differentiation 4E–103 Response to peptide hormone stimulus 4E–80 f ChIP-Seq: Brd4 Response to insulin stimulus 6E–80 D7 100 kb Generation of precursor metabolites 1E–54 (adipocyte) Regulation of fatty acid oxidation 6E–45 D0 White fat cell differentiation 3E–44 Regulation of mitochondrion organization 4E–40 D2 Muscle structure development 5E–22 D7 Adipogenesis Muscle cell differentiation 4E–16 Myocyte Myocyte Actin cytoskeleton organization 2E–14 Pparg1 (C2C12) Organelle assembly 1E–13 Pparg2 Muscle tissue development 2E–13 Striated muscle tissue development 3E–13 20 kb

D0 D2

Adipogenesis D7 Myocyte Myod

Fig. 3 Cell-type- and differentiation-stage-specific binding of Brd4 on cell identity genes. Adipogenesis and myogenesis were done as in Fig. 2 and Supplementary Fig. 4, respectively. Cells were collected for ChIP-Seq analysis of Brd4. a Venn diagram depicting high-confidence Brd4 binding regions at D0 (preadipocytes), D2 (during adipogenesis), and D7 (adipocytes) of adipogenesis. High-confidence Brd4 binding regions were obtained by selecting overlapping regions from two biological replicates. b Western blot analysis of Brd4 during adipogenesis using nuclear extracts prepared from two independent brown preadipocyte cell lines. c Heat maps of Brd4 binding sites during adipogenesis. d Venn diagram representing Brd4 binding regions in adipocytes and myocytes. e GO analysis of genes associated with Brd4 binding regions at indicated time points and cell types. f ChIP-Seq profiles of Brd4 binding on gene loci encoding PPARγ and MyoD at indicated time points and cell types

Brd4 binding sites due to off-target effect of the antibody, ChIP- biological functions. However, Brd4 moves to adipogenesis- Seq was also done in Brd4 KO cells. To identify high-confidence related genes at D2 and D7 of differentiation (Fig. 3e). In C2C12 Brd4 binding sites, we first removed false-positive signals myocytes, preferred target genes of Brd4 were those involved in obtained from Brd4 KO cells and then selected overlapping peaks muscle cell differentiation. Accordingly, we observed differentia- from two biological replicates. In total, we identified 37,111, tion-stage- and cell-type-specific genomic binding of Brd4 on 13,676, and 27,351 Brd4 binding regions at D0, D2, and D7, Pparg and Myod1 loci, which encode the master adipogenic TF respectively (Fig. 3a). Brd4 protein levels decreased only mildly at PPARγ and myogenic TF MyoD, respectively (Fig. 3f). Together, D2 (Fig. 3b), but the genomic binding of Brd4 redistributed these results indicate cell-type- and differentiation-stage-specific dramatically from D0 to D2 (Fig. 3c). Around 89% of D0 Brd4 genomic binding of Brd4 on cell identity genes. binding regions were lost at D2, although some of the lost regions were re-occupied by Brd4 at D7. We also performed Brd4 ChIP- Brd4 co-localizes with LDTFs on active enhancers. Next, we Seq in C2C12 myocytes. The Brd4 binding regions in adipocytes performed motif analysis of the top 3000 Brd4 binding regions at and C2C12 myocytes were largely non-overlapping (Fig. 3d). each time point and in different cell types (Fig. 4a). In pre- To characterize Brd4-associated genes in different stages and adipocytes (D0), Brd4 binding regions were enriched with motifs cell types, we assigned each Brd4 binding region to the nearest of AP-1 family of TFs Jun, Jdp2, and JunD. This is consistent with annotated gene. Gene ontology (GO) analysis revealed that at D0 the previous finding that Brd4 interacts directly with c-Jun21. before differentiation, Brd4 binds to genes associated with general During (D2) and after (D7) adipogenesis, Brd4 binding regions

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abMotif analysis: Brd4 binding sites ChIP-Seq (D2) Brd4 (13676) D0 (preadipocyte)

Factors Logo P value 5441

JUN 8E–32 4519 JDP2 2E–23 2383 2346 JUND 3E–12 17473 TCF3 8E–10 PPARγ C/EBPs (6733) (25464) D2 (during adipogenesis) c Factors Logo P value Heat maps (D2) Brd4 C/EBPs PPARγ C/EBPα 2E–307 C/EBPβ 6E–277 ATF4 1E–85 PPARγ 4E–43 DBP 7E–42 EBF1 1E–37 NFIC 6E–33 13676 Brd4 binding sites HLF 5E–30 –100 +10 –100 +10 –100 +10 GR 2E–16 Distance from Brd4 binding sites (kb) CREB5 5E–15 CREB1 1E–12 d IP Input Brd4 RbBP5 CDK9 D7 (adipocyte) –+–+–+–+ 4-OHT 250 Factors Logo P value 150 Brd4 C/EBPβ 8E–32 75 RbBP5 C/EBPα 6E–21 50 ATF4 6E–16 * 37 C/EBPβ 2E–13 ELK3 37 PPARγ 7E–13 CDK9

Fig. 4 Genomic co-localization of Brd4 with LDTFs during adipogenesis. a Motif analysis of Brd4 binding regions in adipogenesis. Only TFs that are expressed at the indicated cell stages are presented. b, c Venn diagram b and heat maps c of genomic co-localization of Brd4 with C/EBPs (C/EBPα or β) and PPARγ at D2 of adipogenesis. Published ChIP-Seq data sets for C/EBPα/β and PPARγ were used (GSE74189)24. d Brd4 physically interacts with C/ EBPβ during adipogenesis. SV40T-immortalized Brd4f/f #2;Cre-ER brown preadipocytes were treated with 4-hydroxytamoxifen (4-OHT) to induce the deletion of exon 5 of Brd4 gene. Immunoprecipitation (IP) of Brd4, RbBP5, and CDK9 was done in nuclear extracts prepared at D2 of adipogenesis. The immunoprecipitates were analyzed by western blot using antibodies indicated on the right. The asterisk (*) indicates IgG heavy chain were enriched with motifs of adipogenic TFs such as C/EBPα,C/ co-localized with adipogenic TFs on active enhancers at D2 EBPβ, and PPARγ as well as ATF4, which was recently identified (Supplementary Fig 6). Adipogenic enhancers were defined as as a novel TF that promotes adipogenesis22. ChIP-Seq analyses of active enhancers that are bound by C/EBPs or PPARγ19. C/EBPα, C/EBPβ, and PPARγ at D2 of adipogenesis confirmed Consistent with the genomic distribution, Brd4 was highly the genomic co-occupancy of Brd4 with these adipogenic TFs enriched on adipogenic enhancers at D2 but enriched on (Fig. 4b, c). Particularly, over 50% of the Brd4 binding regions adipogenic promoters, which associate with adipogenic enhan- showed co-occupancy with C/EBPα/β. Consistent with previous cers, at D7 (Fig. 5c). At D2, Brd4 binding was observed on 43.9%, − − reports that Brd4 interacts with C/EBPα/β11,21, we observed a 41.7%, and 78.8% of C/EBP+PPARγ , C/EBP PPARγ+, and C/ physical interaction between Brd4 and C/EBPβ during adipo- EBP+PPARγ+ adipogenic enhancers, respectively (Fig. 5d). genesis (Fig. 4d). In C2C12 myocytes, Brd4 binding regions were Together, these results indicate that Brd4 co-localizes with enriched with motifs of myogenic TF MyoD and its binding LDTFs on active enhancers during adipogenesis. partner TCF3 (Supplementary Fig 5a)23. We also confirmed genomic co-localization of Brd4 with MyoD in C2C12 myocytes LDTFs, MLL3/MLL4, and p300 recruit Brd4 to active enhan- (Supplementary Fig 5b, c). cers. Next, we investigated the mechanisms that recruit Brd4 to Next, we characterized the genomic features of Brd4 binding active enhancers during adipogenesis. Notably, 73.8% (6129/ fi regions. Based on histone modi cations, four types of regulatory 8309) of Brd4 binding sites within active enhancers were occu- fi 19 elements were de ned as described : active enhancer, primed pied by enhancer epigenomic writers MLL4 and/or p300, indi- enhancer (previously described as silent enhancer), active cating a substantial overlap (Fig. 6a). Among the 8309 Brd4+ promoter, and silent promoter (Fig. 5a). Interestingly, Brd4 active enhancers, substantially more of them were co-occupied by binding sites were mainly located on active enhancers at D0 and both epigenomic writers (p300 or MLL4) and LDTFs (C/EBPs or D2 but on active promoters at D7 (Fig. 5b). Consistently, Brd4 PPARγ) than by either alone (Fig. 6b, c).

NATURE COMMUNICATIONS | 8: 2217 | DOI: 10.1038/s41467-017-02403-5 | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02403-5 a (Fig. 6f). Decreased binding of Brd4 in Mll3/Mll4 KO cells could Regulatory element Histone mark be due to reduced binding of LDTFs to enhancers. Indeed, 60% (3725/6158) of C/EBPβ+ MLL4+ active enhancers showed Active enhancer H3K4me1(+) H3K4me3(–) H3K27ac(+) decreased C/EBPβ binding in Mll3/Mll4 KO cells, which Primed enhancer H3K4me1(+) H3K4me3(–) H3K27ac(–) Active promoter ± consequently led to decreases in p300 and Brd4 binding (Fig. 6g). H3K4me3(+) within TSS 1 kb + + Silent promoter H3K4me3(–) within TSS ± 1 kb However, on the remaining 40% (2433/6158) of C/EBPβ MLL4 active enhancers, deletion of Mll3/Mll4 in preadipocytes did not β b affect C/EBP binding but prevented p300 and Brd4 binding at Brd4 genomic distribution D2 of adipogenesis. Deletion of Mll3/Mll4 also reduced p300 and D0 D2 D7 Brd4 binding to enhancers on Pparg gene locus during (37111) (13676) (27351) Active enhancer adipogenesis (Fig. 6h). Together, these results suggest that LDTFs Primed enhancer and epigenomic writers MLL3/MLL4 and p300 coordinate to 3867 11483 10023 6408 1163 7737 Active promoter recruit Brd4 to active enhancers during adipogenesis. Silent promoter 17518 8309 7066 Other Brd4 is required for Pol II binding on active enhancers.We next asked how Brd4 regulates cell identity gene induction during c Average profile of Brd4 adipogenesis. For this purpose, we selected several adipogenic Adipogenic Adipogenic enhancers (e1–e8) on cell identity genes Pparg, Cebpa, and Fabp4, enhancers promoters which are bound by Brd4 during (D2) adipogenesis (Fig. 7a). We 50 40 examined the occupancy of the early adipogenic TF C/EBPβ, 40 30 D0 30 MLL4, MLL3/MLL4-mediated H3K4me1, CBP/p300-mediated 20 D2 20 H3K27ac, Brd4, the MED1 subunit of the Mediator coactivator 10 D7

read count complex, the TBP subunit of the general transcription factor

Normalized 10 26 0 0 (GTF) TFIID , Pol II, and the catalytic subunit CDK9 of the –5 0 +5 –0.5 0 +0.5 positive transcription elongation factor b (p-TEFb)27 on these Distance from regulatory element (kb) enhancers during adipogenesis. We did not observe changes in C/ EBPβ and MLL4 binding as well as H3K4me1 and H3K27ac levels d Heat maps (D2 adipogenic enhancers) on Brd4+ adipogenic enhancers in Brd4 KO cells. However, C/EBPα C/EBPβ PPARγ Brd4 deletion of Brd4 markedly reduced MED1, TBP, Pol II, and CDK9 binding on Brd4+ adipogenic enhancers but not on enhancers near constitutively active genes Arid1a and Jak1 (n1 and n2) (Fig. 7b). Accordingly, deletion of Brd4 decreased eRNA C/EBP+PPARγ– production from Brd4+ adipogenic enhancers (Fig. 7c). These (4991) data suggest a model that sequential binding of LDTFs, epige- 51.7% 99.1% 0% 43.9% nomic writers MLL3/MLL4, and CBP/p300 facilitates Brd4 C/EBP+PPARγ+ binding on active enhancers, which is required for enhancer (1731) 82.1% 97.5% 100% 78.8% binding of Mediator, TFIID, Pol II and p-TEFb, eRNA produc- C/EBP–PPARγ+ tion, and cell identity gene induction during adipogenesis 0% 0% 100% 41.7% (1152) (Fig. 7d). –100 +10 –100 +10 –100 +10 –100 +10 Distance from adipogenic enhancers (kb) Brd4 is largely dispensable for maintaining adipocytes. Term- Fig. 5 Brd4 co-localizes with LDTFs on active enhancers during inally differentiated adipocytes express high levels of cell identity adipogenesis. a Definition of regulatory elements by histone marks. b Pie genes including master adipogenic TFs Pparg and Cebpa.We charts depicting the genomic distributions of Brd4 binding regions during asked whether Brd4 is required to maintain adipocyte gene adipogenesis. c Average binding profiles of Brd4 around the center of expression. To this end, we crossed Brd4f/f mice with Adipoq-Cre adipogenic enhancers and promoters. Adipogenic enhancers were defined mice to generate adipocyte-specific Brd4 KO mice28. Deletion of as active enhancers bound with C/EBPα, C/EBPβ,orPPARγ at D2 of Brd4 was successful in adipose tissues (Fig. 8a, b). However, we adipogenesis as described previously19. Adipogenic promoters were defined did not observe any discernable differences in adipose tissue mass as promoters that are closest to adipogenic enhancers. d Brd4 co-localizes (Fig. 8c, d) or the expression of adipocyte identity genes Pparg, with LDTFs on active enhancers during adipogenesis. The binding profiles of Cebpa, Fabp4, or BAT marker gene Ucp1 (Fig. 8e). RNA-Seq C/EBPα, C/EBPβ,PPARγ, and Brd4 on the three types of adipogenic analysis of BAT and epididymal white adipose tissue (eWAT) enhancers (C/EBP+PPARγ−, C/EBP−PPARγ+, and C/EBP+PPARγ+) are from Brd4f/f;Adipoq-Cre mice confirmed that the deletion of Brd4 shown in heat maps in adipose tissues does not affect adipocyte and BAT-enriched gene expression (Fig. 8f, g). Our data suggest that while Brd4 is essential for adipose tissue development, it is largely dispensable Since MLL3/MLL4 facilitate CBP/p300 binding to enhancers for the maintenance of adipose tissues and related gene during cell differentiation24,25 and CBP/p300 facilitate Brd4 expression. binding to active enhancers11, we hypothesized that MLL3/MLL4 are required for Brd4 recruitment to enhancers during differ- JQ1 blocks PPARγ-stimulated adipogenesis. Since Brd4 is entiation. To test this hypothesis, we performed Brd4 ChIP-Seq in essential for the induction of PPARγ in the early stage of adi- Mll3/Mll4 double KO cells at D2 of adipogenesis. Although pogenesis, we tested whether forced expression of PPARγ could deletion of Mll3/Mll4 did not affect Brd4 protein levels (Fig. 6d), rescue adipogenesis in Brd4 KO cells. Indeed, adipogenesis defects it led to the loss of 90.3% of total Brd4 binding sites at D2 in Brd4 KO cells could be rescued by retroviral vector-mediated (Fig. 6e). Consistently, we observed a marked decrease in Brd4 expression of ectopic PPARγ (Fig. 9a, b). Interestingly, the binding levels on MLL4+ active enhancers in Mll3/Mll4 KO cells expression levels of Brd2 and Brd3 increased in Brd4 KO cells,

6 NATURE COMMUNICATIONS | 8: 2217 | DOI: 10.1038/s41467-017-02403-5 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02403-5 ARTICLE

a ChIP-Seq (D2, AEs) + + Brd4+ AE (8309) bcBrd4 AE Brd4 AE 60 50 2180 1552 40 40 30 3675 20 20 5796

2990 Fraction (%) Fraction (%) 10 MLL4+ AE p300+ AE 0 0 p300 + – + MLL4 + – + (8814) (14013) C/EBPs / PPARγ –++ C/EBPs / PPARγ –++

Mll3–/– Mll4f/f cells d g D0 D2 C/EBPβ p300 Brd4 MLL4 GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre 250 Brd4 I (2433) 94.6% 54.0%87.2% 19.3% 75 RbBP5 II (3725) 82.7% 13.7% 75.4% 4.8% e ChIP-Seq: Brd4 (D2) –50–5+5 +5 0–5+50–5+50 –50–5 +5 0–5+5+5 0 GFP Cre Distance from C/EBPβ+ MLL4+ binding AEs at D2 (kb) (13257) (2334) h 11968 D0 (90.3%) 1045 1289

MLL4 D2 GFP D0 Cre f Average plot (D2)

Brd4 p300 GFP D2 GFP Cre 20 Cre GFP D0 10 Cre read count Normalized

Brd4 GFP 0 D2 –2.50 +2.5 Cre Distance from + MLL4 AEs (kb) PPAR1 PPAR2

Fig. 6 LDTFs, MLL3/MLL4, and p300 coordinate to recruit Brd4 to active enhancers. a Venn diagram of co-localization of Brd4, MLL4, and p300 on active enhancers (AEs) at D2 of adipogenesis. b, c Brd4 binding is highly enriched on AEs that are bound by both LDTFs (C/EBPs or PPARγ) and p300 b or MLL4 c during adipogenesis. d–h MLL3/MLL4 are required for Brd4 binding on AEs during adipogenesis. Immortalized Mll3−/− Mll4f/f preadipocytes were infected with adenoviral GFP or Cre as described previously19, followed by adipogenesis assay. Cells were collected before (D0) and during (D2) adipogenesis for ChIP-Seq of Brd4. d Deletion of MLL3/MLL4 does not affect Brd4 protein levels. e Venn diagram representing Brd4 binding regions in GFP- and Cre-infected Mll3−/− Mll4f/f cells at D2 of adipogenesis. f Average profile of Brd4 binding on MLL4+ AEs at D2. g Heat maps around C/EBPβ+ MLL4+ AEs at D2. AEs were classified into two groups: unchanged (group I) and decreased (group II) C/EBPβ binding in Mll3/Mll4 double KO cells. h Genome browser view of p300 and Brd4 binding on Pparg locus in GFP- and Cre-infected Mll3−/− Mll4f/f cells suggesting a functional redundancy among BET family proteins Discussion (Fig. 9b). Inhibiting BET proteins by JQ1 treatment completely Using two independently developed Brd4 conditional KO mice blocked PPARγ-stimulated adipogenesis (Fig. 9a, b). These results and derived cell lines, we demonstrate that Brd4 is essential for indicate that Brd4 is the major functional BET protein before the adipogenesis and myogenesis in culture and in vivo. Using RNA- induction of PPARγ and suggest that Brd4 is functionally Seq, we show Brd4 promotes cell-type-specific gene expression redundant with Brd2/Brd3 in promoting adipocyte gene expres- during cell differentiation. Using ChIP-Seq, we show Brd4 pre- sion downstream of PPARγ. Consistent with these results, inhi- dominantly binds to active enhancers during adipogenesis but biting BET proteins by JQ1 treatment inhibited the synthetic preferentially on active promoters after adipogenesis. Further, PPARγ ligand rosiglitazone (Rosi)-induced expression of PPARγ enhancer epigenomic writers MLL3/MLL4 are required for Brd4 target genes Cebpa, Adipoq, and Fabp4 in undifferentiated pre- binding to active enhancers during adipogenesis. Finally, Brd4 adipocytes expressing ectopic PPARγ (Fig. 7c), indicating that facilitates enhancer binding of Mediator, TFIID, Pol II, and p- BET family proteins are required for PPARγ target gene TEFb, and eRNA transcription during adipogenesis. Our findings expression. identify Brd4 as an enhancer epigenomic reader that connects active enhancers with cell identity gene induction during

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a c qRT-PCR 50 kb e1 e2e3e4 eRNAs 45 Brd4 30 Pparg1 Pparg2 15 10 kb e5 e6 0 Expression (fold) Brd4 e1 e2 e3 e4 e5 e6 e7 e8 n1 n2

Cebpa d Enhancer priming by MLL3/MLL4 10 kb e7 e8 MLL3/4 Brd4 α β

me me γ me me Off Fabp4 PPAR C/EBP C/EBP TSS b ChIP-qPCR C/EBPβ Brd4 Enhancer activation by CBP/p300 2.0 0.6 GFP D0 1.5 p300/CBP MLL3/4 GFP D2 0.4 Cre D0 α β

1.0 γ Cre D2 ac ac 0.5 0.2 Off PPAR C/EBP 0 0 C/EBP MLL4 MED1 0.25 0.9 TSS 0.20 0.15 0.6 Brd4 binding to active enhancers 0.10 0.3 0.05 p300 MLL3/4 0 0 Brd4 H3K4me1 TBP 7.5 0.25 Off

% of input 0.20 5.0 0.15 TSS 2.5 0.10 0.05 0 0 Recruitment of mediator, GTFs, Pol II, & eRNA production H3K27ac Pol II 7.5 0.25 0.20 p300 MLL3/4 5.0 0.15 Brd4 0.10 On 2.5 GTFs 0.05 Med & Pol II 0 0 CDK9 TSS e1 e2 e3 e4 e5 e6 e7 e8 n1 n2 0.15 0.10

0.05 Adipocyte gene expression me H3K4me1 0 e1 e2 e3 e4 e5 e6 e7 e8n1 n2 ac H3K27ac

Fig. 7 Brd4 is required for Pol II binding on active enhancers during adipogenesis. a Schematic of genomic locations of Brd4+ enhancers (e1–e8) on Pparg, Cebpa, and Fabp4 loci. b Brd4 is dispensable for C/EBPβ, MLL4, H3K4me1, and H3K27ac enrichment, but is essential for MED1, TBP, Pol II, and CDK9 binding, on adipogenic enhancers. ChIP-qPCR analyses of indicated factors are shown on adipogenic enhancers (e1–e8). Enhancers of constitutively expressed genes Arid1a (n1) and Jak1 (n2) were chosen as negative control regions. c Brd4 is required for eRNA transcription on adipogenic enhancers. The data are presented as means ± SD. Three technical replicates from a single experiment were used. d Model depicting that sequential actions of LDTFs, H3K4 mono-methyltransferases MLL3/MLL4, H3K27 acetyltransferases CBP/p300, epigenomic reader Brd4, transcription coactivator Mediator, and Pol II on enhancers control cell identity gene expression during differentiation differentiation. Together with previous findings in the literature, leads to the compromised innate immune response34. By crossing our data suggest a model in which sequential actions of LDTFs, Brd4f/f with Myf5-Cre mice, we demonstrate that Brd4 is required H3K4 mono-methyltransferases MLL3/MLL4, H3K27 acetyl- for adipose tissue and muscle development in vivo. By knocking transferases CBP/p300, epigenomic reader Brd4, transcription down Brd4 in 3T3-L1 white preadipocytes and C2C12 myoblasts coactivator Mediator, and Pol II transcription machinery on and knocking out Brd4 in brown preadipocytes, we confirmed enhancers control cell identity gene expression during that the essential role of Brd4 in adipogenesis and myogenesis is differentiation. cell-autonomous. Our finding on Brd4 in adipogenesis is con- Brd4 inhibitors are promising drug candidates for treating sistent with a previous report that JQ1 inhibits differentiation of cancers and other diseases29,30. However, few studies have looked mesenchymal C3H10T1/2 cells toward adipocytes in culture35. at the role of Brd4 in cell differentiation and animal development, JQ1 treatment cannot distinguish functional roles of BET proteins except that Brd4 is required for the differentiation of erythroid in adipogenesis. Our genetic study distinguishes the roles of BET and osteoblast in cell culture31,32, that inducible knockdown of family members in adipogenesis and indicates that Brd4 is the Brd4 in mice results in skin hyperplasia and loss of cell diversity major BET protein controlling PPARγ induction in the early in intestine33, and that myeloid lineage-specific deletion of Brd4 phase of adipogenesis while the functionally redundant Brd2,

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Brd4f/f × adipoq-Cre mice

a f/f bdc Brd4 (f/f) f/f cKO Brd4f/f;adipoq-Cre (cKO) BAT eWAT BAT iWAT eWAT f/f cKO f/f cKO BAT 0.3 2.0 2.5 1.5 p = 0.86 p = 0.49 p = 0.95 Brd4 1212 1212 1.5 2.0 0.2 1.5 1.0 250 1.0 Brd4 iWAT 1.0 0.1 0.5 0.5 0.5 75 RbBP5

mRNA (fold) eWAT 0 0 0 % of body weight f/f cKO f/f cKO f/f cKO 0 BAT eWAT

e BAT eWAT g BAT eWAT Pparg Cebpa Fabp4 Ucp1 Pparg Cebpa Fabp4 1.5 1.5 1.5 1.5 1.5 1.5 1.5 f/f cKOf/f cKO

Scd1 Adipogenesis / BAT marker genes 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Fabp4 Lpl Dgat2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Lipe

mRNA (fold) Scd3 0 0 0 0 0 0 0 f/f cKO Adipoq f/f cKO f/f cKO f/f cKO f/f cKO f/f cKO f/f cKO Cebpa Adipor2 f Cox5b f/f Scd2 Cebpb BAT cKO Lpin1 Dgat1 f/f Pparg cKO Adrb3

eWAT Adipor1 Brd4 Pex11a Add1

11 10 9 8 76 5 4 3 Log Ebf2 10

Ucp1 2 (RPKM+0.01) Cidea 5 Cox7a1 Pdk4 0 Ppara Elovl3 –5 Ppargc1a –10 Prdm16

Fig. 8 Brd4 is dispensable for the maintenance of cell identity gene expression in adipocytes. Brd4f/f mice were crossed with Adipoq-Cre to generate adipocyte-specific Brd4 KO mice. a qRT-PCR confirmation of Adipoq-Cre-mediated Brd4 deletion in mouse adipose tissues (n = 6 per group). b Western blot analysis of Brd4 in BAT and eWAT. c Representative morphology of mouse adipose tissues. d Adipose tissue mass was normalized to body weight (n = 6 per group). Statistical comparison between groups was performed using Student’s t -test. The data are presented as means ± SEM. e qRT-PCR of Pparg, Cebpa, Fabp4, and Ucp1 expression in adipose tissues (n = 4 per group). Quantitative PCR data in this figure are presented as means ± SEM. f, g Deletion of Brd4 does not affect adipocyte gene expression in adipose tissues. Equal amounts of total RNA from BAT and eWAT of four 12-week-old male mice were combined for RNA-Seq analysis. f Genome browser view of RNA-Seq analysis on Brd4 gene locus. The targeted exon 3 is highlighted in red box. g Heat map of RNA-Seq values of adipogenesis/BAT marker genes

Brd3, and Brd4 control the induction of PPARγ downstream stem cell differentiation24,25. It has been shown that hemato- adipocyte genes. poietic LDTFs and CBP/p300 facilitate Brd4 recruitment to active Brd4 localizes on both active promoters and active enhancers enhancers in leukemia cells11. Similarly, our data show that adi- in human and mouse tumor cells10,11. In this study, we show pogenic TFs and p300 cooperatively recruit Brd4 to active dynamic changes of Brd4 binding regions at various stages of enhancers during adipogenesis. Furthermore, we demonstrate differentiation. In our adipogenesis model system, Brd4 mainly that MLL3/MLL4 are critical for Brd4 binding on enhancers. localizes on active enhancers that are associated with cell identity Consistent with our data, a recent study showed that Brd4 is genes induced during differentiation. After differentiation, Brd4 required for binding of Mediator and CDK9 on enhancers binding on active enhancers largely remains but with reduced including the Myc super-enhancer in mouse acute myeloid leu- binding intensity. Interestingly, Brd4 binds to ~75% of all active kemia (AML cells)13. Brd4 may regulate Mediator binding to promoters in differentiated cells, while only ~8% of all active enhancers through its physical interaction with Mediator com- promoters are occupied by Brd4 during differentiation. These plex27. On the other hand, since PPARγ physically associates with results suggest distinct functions of Brd4 during and after cell Mediator complex as well36, it is also possible that decreased differentiation. In differentiating cells, Brd4 localization on active Mediator binding in Brd4 KO cells is due to the decreased enhancers controls cell identity gene induction necessary for expression of PPARγ, a direct target of Brd4. Physical and terminal differentiation. The role of Brd4 on active promoters in functional association between Brd4 and CDK9, a component of terminally differentiated cells remains to be understood. p-TEFb, has been well documented27,37. Our data suggest that LDTFs bind to cell-type-speciﬁc enhancers and recruit H3K4 Brd4 is a molecular bridge between cell-type-speciﬁc enhancers mono-methyltransferases MLL3/MLL419. MLL3/MLL4 are and general transcription machinery. Taken together, we propose required for enhancer binding of H3K27 acetyltransferases CBP/ a model that adipocyte gene expression is induced by sequential p300 and enhancer activation during adipogenesis and embryonic actions of LDTFs C/EBPα, C/EBPβ, and PPARγ, enhancer

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were purchased from Sigma. Anti-RbBP5 (A300–109A), anti-Brd4 a GFP Cre (A301–985A100), and anti-MED1 (A300–793A) were from Bethyl Laboratories. JQ1 – – – + Anti-C/EBPα (sc-61X), anti-C/EBPβ (sc-150X), anti-PPARγ (sc-7196X), and anti- PPARγ – – + + p300 (sc-585X) were from Santa Cruz Biotechnology. Anti-H3K4me1 (ab8895), anti-H3K4me2 (ab7766), and anti-H3K27ac (ab4729) were from Abcam. Anti-Pol II (17-672) was from Millipore. For western blot analysis, all antibodies were − diluted to 1 μgml 1. Uncropped blots are available in the Supplementary Figure 7.

Generation of mouse strains. Two Brd4 conditional KO mouse lines were used in this study. In the first Brd4 conditional KO mouse line (Brd4f/f), the first coding exon of Brd4 gene, exon 3, is flanked by loxP sites. Genotyping the Brd4 alleles, PCR was done using the following primers: 5′-GCCTA- GATCAGTGCCTCCATTG-3′ and 5′-ACTGGAACTACATGGCAGCCTG-3′. PCR amplified 244 bp from the wild-type and 344 bp from the floxed allele. Brd4f/f b 4 1.5 mice were crossed with Myf5-Cre (Jackson no. 007893, C57BL/6 J and 129S4/ Cebpa Fabp4 SvJaeSor mixed background) or Adipoq-Cre (C57BL/6 background)28 to generate 3 1.0 Brd4f/f;Myf5-Cre or Brd4f/f;Adipoq-Cre mice. For characterization of Brd4f/f; 2 Adipoq-Cre mice, we used six 12-week-old male mice per genotype. Animals were 0.5 not randomized and the researchers were not blinded during the experiment and 1 when assessing the outcome. No animals were excluded from the analysis. f/f #2 0 0 To establish the second Brd4 conditional KO mouse line (Brd4 ), the heterozygous conditional Brd4 gene trap mice (Brd4tm1a(EUCOMM)Wtsi) were 1.5 3 3 obtained from the KOMP2 program at Baylor College of Medicine and crossed with Brd4 Brd2 Brd3 FLP1 mice (Jackson no. 003946) to delete the neomycin cassette. In the resulting 1.0 2 f/f #2 fl mRNA levels (fold) 2 Brd4 mice, the exon 5 of Brd4 gene is anked by loxP sites. Genotyping of the Brd4 alleles was done using the following primers: 5'-GGACATGGTGACAGAG 0.5 1 1 TGG-3' and 5'-TCAAATGAATTCACTAGAACTAC-3'. PCR amplified 168 bp from the wild-type and 284 bp from the floxed allele. Brd4f/f #2 mice were crossed 0 0 0 with Cre-ER (Jackson no. 008463) to generate Brd4f/f #2;Cre-ER mice. Cre – + + +–+ + + – + + + All mouse experiments were performed in accordance with the NIH Guide for JQ1 – – – + – – – + – – – + the Care and Use of Laboratory Animals and approved by the Animal Care and PPARγ – – ++ – – ++ – – ++ Use Committee of NIDDK, NIH. c 6 150 Histology and immunohistochemistry. E18.5 embryos were isolated by Cesarean Pparg Cebpa section, fixed in 4% paraformaldehyde, dehydrated in a methanol series, and 4 100 embedded in paraffin for sectioning. Paraffin sections were stained with routine H&E or subjected to immunohistochemistry using anti-Myosin (MF20; Develop- 2 50 mental Studies Hybridoma Bank, 1:20 dilution) and anti-UCP1 (ab10983; Abcam, 1:400 dilution) antibodies19. 0 0 9000 200 Cell culture and differentiation assays. Primary brown preadipocytes were Adipoq Fabp4 f/f f/f #2 150 isolated from interscapular BAT of newborn Brd4 , Brd4 ;Cre-ER and mRNA levels (fold) 6000 immortalized by infecting retroviruses expressing SV40T38. Adipogenesis of 100 immortalized brown preadipocytes was induced with DMEM supplemented with − 3000 μ μ 1 50 10% fetal bovine serum (FBS), 0.02 M insulin, 1 nM T3, 0.5 mM IBMX, 2 gml DEX, and 0.125 mM indomethacin for 2 days39. After this period, the culture 0 0 medium was supplemented with FBS, insulin, and T3 only. 3T3-L1 cells were from JQ1 – – – + – – – + Daniel Lane. PPARγ – + + + – + + + C2C12 myoblasts were purchased from ATCC and cultured in growth medium Rosi – – + + – – + + of DMEM containing 15% FBS. Myogenesis was induced by replacing growth medium to DMEM containing 2% horse serum when cells were ~70% confluent. Fig. 9 JQ1, but not the single KO of Brd4, blocks PPARγ-stimulated Before changing the medium, cells were washed with plain DMEM twice. adipogenesis. a, b Brd4f/f brown preadipocytes were infected with adenoviral GFP or Cre. Cre-infected cells were further infected with qRT-PCR. Total RNA was extracted using TRIzol (Invitrogen) and reverse tran- retroviruses expressing vector alone or PPARγ. After hygromycin selection, scribed using ProtoScript II first-strand cDNA synthesis kit (NEB), following ’ cells were induced for adipogenesis assay in the presence or absence of manufacturer s instructions. qRT-PCR of Brd4 exon 3 was done using SYBR green primers: forward 5′-CCCAGAGACCTCCAACCCTAA-3′ and reverse 5′- 0.5 μM BET inhibitor JQ1. a Oil red O staining at D7 of adipogenesis. Scale AACTGGTGTTTCCATAGTGTCTTGAG-3′. qRT-PCR of Brd4 exon 5 was done bars = 15 μm. b qRT-PCR of Cebpa, Fabp4, Brd4, Brd2, and Brd3 at D7 of using the primers: forward 5′-TGACATCGTCTTAATGGCAGAAG-3′ and reverse adipogenesis. c BET family proteins are required for ligand-induced PPARγ 5′-CCTTTTGCCTGGACTATCATGAT-3′. target gene expression. Subconfluent Brd4f/f preadipocytes expressing ectopic PPARγ were pretreated with JQ1 (0.5 μM) for 3 h followed by ChIP-Seq and RNA-Seq. For ChIP-Seq analysis, formaldehyde was added directly fi treatment with 1 μM rosiglitazone (Rosi) for 24 h in the presence of JQ1. to cell culture medium to a nal concentration of 2%. After 10 min of incubation at ± room temperature, glycine was added to a concentration of 125 mM to quench The data are presented as means SD. Three technical replicates from a crosslinking reaction. Approximately 2 × 107 cells were washed with 20 ml cold single experiment were used PBS in culture dish twice and scraped off in 10 ml Farnham lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40, supplemented with protease inhibitors), then pelleted by centrifugation at 3000×g for 5 min at 4 °C. Cell pellet was resus- epigenomic writers MLL3/MLL4 and CBP/p300, enhancer epi- pended in 10 ml lysis buffer and pelleted again to remove cytosolic proteins. genomic reader Brd4, Mediator, and Pol II transcription Resulting nuclear pellet was sonicated in 2 ml TE buffer (10 mM Tris-Cl, pH 8.0, machinery on enhancers (Fig. 7d). 1 mM EDTA, supplemented with protease inhibitors) to achieve DNA fragments of 200–500 bp. Detergents were added to digested chromatin fractions to make 1× RIPA buffer (10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium Methods deoxycholate, 1% triton X-100). After centrifugation, supernatant was collected in a Plasmids and antibodies. The lentiviral shRNA plasmids pLKO.1 targeting mouse new tube. For each ChIP, 8–10 μg antibodies were pre-incubated with 50 μl Brd4 (clone IDs TRCN0000311976, TRCN0000088480, and TRCN0000088481) Dynabeads Protein A (Life Technologies) in 1 ml PBS overnight at 4 °C under

10 NATURE COMMUNICATIONS | 8: 2217 | DOI: 10.1038/s41467-017-02403-5 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02403-5 ARTICLE gentle rotation. Next day, 1 ml chromatin from 1 × 107 cells was mixed with 14. Kanno, T. et al. BRD4 assists elongation of both coding and enhancer RNAs by antibody-beads complex and incubated overnight at 4 °C with gentle rotation. interacting with acetylated histones. Nat. Struct. Mol. Biol. 21, 1047–1057 Chromatin immunoprecipitates were washed twice with 1 ml RIPA buffer, twice (2014). with 1 ml RIPA containing 300 mM NaCl, twice with 1 ml LiCl buffer (50 mM 15. Lefterova, M. I., Haakonsson, A. K., Lazar, M. A. & Mandrup, S. PPARgamma Tris-Cl, pH 7.5, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and twice and the global map of adipogenesis and beyond. Trends Endocrinol. Metab. 25, with 1 ml TE buffer. Samples were reverse-crosslinked in 100 μl elution buffer (1% 293–302 (2014). SDS, 0.1 M NaHCO3, and 100 μg proteinase K) overnight at 65 °C. DNA was 16. Rosen, E. D. & MacDougald, O. A. Adipocyte differentiation from the inside purified by QIAquick PCR Purification Kit (QIAGEN) and quantified using Qubit out. Nat. Rev. Mol. Cell Biol. 7, 885–896 (2006). fi 19,24 dsDNA HS Assay Kit (Thermo Fisher Scienti c) . 17. Ge, K. Epigenetic regulation of adipogenesis by histone methylation. Biochim. fi fi For RNA-Seq, mRNAs were puri ed using Dynabeads mRNA Puri cation Kit Biophys. Acta 1819, 727–732 (2012). (Invitrogen), and then they were used to synthesize double-stranded cDNAs using 18. Berkes, C. A. & Tapscott, S. J. MyoD and the transcriptional control of SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). Sequencing myogenesis. Semin. Cell Dev. Biol. 16, 585–595 (2005). libraries were constructed using NEBNext Ultra II DNA Library Prep Kit for 19. Lee, J. E. et al. H3K4 mono- and di-methyltransferase MLL4 is required for Illumina (NEB). All ChIP-Seq and RNA-Seq samples were sequenced on the enhancer activation during cell differentiation. eLife 2, e01503 (2013). Illumina HiSeq 2500. 20. Seale, P. et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature 454, 961 (2008). Computational analysis. To identify Brd4 binding regions, we used ‘SICER’ 21. Wu, S. Y., Lee, A. Y., Lai, H. T., Zhang, H. & Chiang, C. M. Phospho switch method with a window size of 50 bp and a gap size of 50 bp40. To eliminate non- triggers Brd4 chromatin binding and activator recruitment for gene-specific specific binding of Brd4 antibody, we compared Brd4 ChIP-Seq data from Brd4 KO targeting. Mol. Cell 49, 843–857 (2013). cells with that from two biological replicates of control (Pparγf/f) cells24, and kept 22. Cohen, D. M. et al. ATF4 licenses C/EBPbeta activity in human mesenchymal only the identified Brd4 binding regions with enrichment level significantly higher stem cells primed for adipogenesis. eLife 4, e06821 (2015). in control cells than in the Brd4 KO cells, with an estimated false discovery rate 23. Lassar, A. B. et al. Functional activity of myogenic HLH proteins requires −3 (FDR) of <10 . Then, we chose only the overlapping Brd4 binding regions from hetero-oligomerization with E12/E47-like proteins in vivo. Cell 66, 305–315 −/− f/f two biological replicates. For Brd4 ChIP-Seq data in Mll3 Mll4 cells, an FDR of (1991). < −10 fi fi 10 was used to nd the high-con dence ChIP-enriched regions. Other pub- 24. Lai, B. et al. MLL3/MLL4 are required for CBP/p300 binding on enhancers and lished ChIP-Seq data sets were downloaded (GSE74189, GSE50466, and 19,24,41 super-enhancer formation in brown adipogenesis. Nucleic Acids Res. 45, GSE44824) . 6388–6403 (2017). For motif analysis of Brd4 binding regions (Fig. 4a), we used SeqPos motif tool 25. Wang, C. et al. Enhancer priming by H3K4 methyltransferase MLL4 controls in Galaxy Cistrome (http://cistrome.org/ap/root)42. The algorithm used in this – 43 cell fate transition. Proc. Natl Acad. Sci. USA 113, 11871 11876 (2016). motif tool is previously described in detail . We selected the top 3000 Brd4 26. Thomas, M. C. & Chiang, C. M. The general transcription machinery and binding regions to screen enriched TF motifs at each time point. general cofactors. Crit. Rev. Biochem. Mol. Biol. 41, 105–178 (2006). 27. Jang, M. K. et al. The bromodomain protein Brd4 is a positive regulatory Data availability. All data sets described in the paper have been deposited in NCBI component of P-TEFb and stimulates RNA polymerase II-dependent Gene Expression Omnibus under accession number GSE99101. transcription. Mol. Cell 19, 523–534 (2005). 28. Wang, Z. V., Deng, Y., Wang, Q. A., Sun, K. & Scherer, P. E. 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NATURE COMMUNICATIONS | 8: 2217 | DOI: 10.1038/s41467-017-02403-5 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02403-5

Taitague for technical support, and NIDDK Genomics Core for sequencing. This work Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in was supported by the Intramural Research Program of NIDDK, NIH to KG. published maps and institutional afﬁliations.

Author contributions J-E.L. and K.G. conceived and designed the experiments. J-E.L., Y-K.P., S.P., and Y.J. Open Access This article is licensed under a Creative Commons performed the experiments. J-E.L., Y-K.P., S.P., Y.J., N.W., B.L., W.P., and K.G. analyzed Attribution 4.0 International License, which permits use, sharing, the data. A.D. and K.O. generated Brd4f/f mice. J-E.L., N.W., B.L., and W.P. performed adaptation, distribution and reproduction in any medium or format, as long as you give computational analyses. J-E.L., S.P., and K.G. wrote the manuscript. K.G. supervised all appropriate credit to the original author(s) and the source, provide a link to the Creative the experiments. Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless Additional information indicated otherwise in a credit line to the material. If material is not included in the Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- article’s Creative Commons license and your intended use is not permitted by statutory 017-02403-5. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Competing interests: The authors declare that they have no competing ﬁnancial licenses/by/4.0/. interests.

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