BET bromodomain proteins regulate enhancer function during adipogenesis

Abstract

Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARγ and C/EBPα, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARγ and C/EBPα expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARγ and C/EBPα. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation. Inhibition of the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins impedes BRD4 occupancy at these de novo enhancers and disrupts transcription of Pparg and Cebpa, thereby blocking adipogenesis. Furthermore, silencing of these BRD4-occupied distal regulatory elements at the Pparg locus by CRISPRi demonstrates a critical role for these enhancers in the control of Pparg gene expression and adipogenesis in 3T3L1s. Together, these data establish BET bromodomain proteins as time- and context-dependent coactivators of the adipocyte cell state transition.

Keywords: BET bromodomain; adipogenesis; chromatin; coactivator; transcription.

Fig. 1.

BET bromodomain proteins control adipocyte differentiation. (A) Photomicrographs of oil red O stained L1 adipocytes after induction of differentiation by DMI (day 8) ± structurally distinct BET bromodomain inhibitors (JQ1 at 500 nM; I-BET, I-BET-151, and PFI-1 at 1 μM). (B and C) Heatmaps of expression of proadipogenic, master regulators (B), and mature adipocyte genes (C) at D0 and D4 of differentiation ± JQ1 (500 nM). (D) Photomicrographs of oil red O staining after differentiation of cells isolated from the stromal vascular fraction of mouse s.c. adipose tissue ± JQ1. Data represent mean ± SEM. The statistical significance of the difference in expression between vehicle (VEH) and JQ1 was determined using a two-tailed t test. (Magnification: 4×.)

Fig. 2.

BETs control expression of the master TFs Pparg and Cebpa. (A and B) Photomicrographs oil red O stained L1s (A) or C3H10T1/2 mesenchymal stem cells (B) after induction of differentiation ± JQ1 (500 nM). JQ1 was added to differentiation medium on indicated days. Staining was on D6 for L1 and D4 for 10T1/2. (Magnification: 4×.) (C and D) Bar plots of fold change in Pparg and Cebpa mRNA levels in 3T3L1 cells and 10T1/2 cells from A and B. ns, not significant. (E) Line plots of fold change in mRNA of early response TFs involved in adipogenesis measured at 1, 6, 24, 48, and 96 h following induction of differentiation ± JQ1 (500 nM). (F) Oil red O staining of L1 adipocytes 8 d after induction of differentiation in the presence or absence of rosiglitazone (1 μM) ± JQ1 (500 nM). (Magnification: 10×.) Data represent mean ± SEM. The statistical significance of the difference in expression between vehicle (VEH) and JQ1 at each time point in C and D was determined using one-way ANOVA with post hoc (Dunnett’s) multiple comparison tests of the means. **P < 0.01, ***P < 0.001. For E, two-way ANOVA was performed with ***P < 0.001 for interaction comparing time x treatment groups.

Fig. 3.

Brd4 is dynamically redistributed to proadipogenic enhancers. (A and B) Plots of enhancers in preadipocytes (D0; A) and adipocytes (D2; B) ranked by increasing BRD4 signal in units of reads per million (rpm). Enhancers are defined as regions of BRD4 ChIP-Seq binding not contained in promoters. The cutoff discriminating typical enhancers from super-enhancers is shown as a dashed line. (C) Horizontal bar plot of all genomic regions containing a SE in preadipocytes (D0) or differentiating adipocytes (D2) ranked by log₂ change in BRD4 signal. The x axis shows the log₂ fold change in BRD4 signal. Change in BRD4 levels at SEs are colored by intensity of change (green to red). (D) Horizontal bar plot showing the ratio of TF motif density between gained (D2) and lost (D0) super-enhancers. Twenty-one TFs are displayed whose motifs occur more frequently than expected based on dinucleotide background model. The motifs are ranked by log₂ fold change in density between D2 gained and D0 lost super-enhancers. (E) Consensus sequences of C/EBPβ (Top) and NR3C1 (Bottom) DNA binding motifs. (F and G) Scatter plot of C/EBPβ versus BRD4 binding signals (F) or C/EBPδ versus BRD4 signals (G) in L1 cells on D2 of differentiation. (H and I) Gene tracks of ChIP-Seq signal (rpm/bp) for BRD4, H3K27ac, C/EBPβ, and PPARγ at the Pparg locus (H) or Ptn (I) locus in 3T3L1 cells on D0 (Upper) or D2 (Lower) of differentiation.

Fig. 4.

BRD4 binding sites regulate Pparg expression and adipogenesis. (A) Gene track of ChIP-Seq signal of BRD4 on D2 of L1 differentiation at the upstream and intragenic Pparg super-enhancer regions (Upper) and bar plot of Pparg mRNA expression (D4 of L1 differentiation) in cells stably expressing dCas9-KRAB alone or with gRNAs targeting individual enhancer constituent sites (E1–E6). (Lower) Three unique gRNAs per enhancer site were used in separate cell lines. (B) Oil red O staining of cells (D7) treated as in A. The statistical significance of the difference in expression between individual gRNA and dCas9-KRAB was determined using two-tailed t test. (Magnification: 4×.) *P < 0.05; ***P < 0.001.

Fig. 5.

Transcription of adipogenic master regulatory factors is BET bromodomain-dependent. (A) Scatter plot of RNA Pol II ChIP-Seq signal at gene bodies of all actively transcribed genes in L1 cells on D2 of differentiation versus D2 + JQ1 (500 nM). (B) Gene tracks of ChIP-Seq signal (rpm/bp) for BRD4 (D2 versus D2 + JQ1 of differentiation) and RNA Pol II on D0 (Top), D2 (Middle), and D2 + JQ1 (500 nM, Bottom) of differentiation at the Pparg (B) locus in L1 cells.