Fig 2: DPF3 regulates the chromatin state at brown fat-specific genes. (A) FAIRE-qPCR analysis of control and DPF3-depleted mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. The insulin and ß-globin promoters serve as negative controls; enrichment was analyzed as percentage of input. (B,C) ChIP-qPCR analysis of BRG1 (B) and EBF2 (C) binding in brown adipocytes ± iso; chromatin enrichment was analyzed as percentage of input recovery and normalized to 18S percentage of input to produce a fold enrichment. The insulin promoter served as a negative control. All data show mean ± SD. n = 3. Two-way ANOVA with Holm-Šídák multiple tests correction comparing shScr versus shDpf3 under basal or iso-stimulated conditions, (*) P < 0.05; (**) P < 0.01.

Fig 3: EBF2 directly regulates Dpf3 expression via an intronic enhancer. (A) ChIP-seq profiles at Dpf3 in RPM for EBF2 (dark blue), H3K27ac (red), RNA Pol II (light blue), and PPAR? (gray) in Ebf2 wild-type and knockout BAT. (B) ChIP-qPCR analysis of EBF2 binding at the Dpf3 +20-kb site over the course of brown adipocyte differentiation. The insulin promoter served as a negative control. Mean ± SD. n = 3. (C) RT-qPCR analysis of Dpf3 expression over the course of brown adipocyte differentiation. Mean ± SD. n = 3. (D) CRISPR–Cas9-mediated genomic editing strategy at the EBF motif in the Dpf3 +20-kb enhancer. (E) ChIP-qPCR for EBF2 in control (Rosa26) and EBF gRNA-expressing pooled brown adipocytes. The insulin promoter served as a negative control. Mean ± SD. n = 3. Two-sample Student's t-test, (**) P < 0.01. (F) Gene expression analysis in control and EBF gRNA-expressing brown adipocytes. Mean ± SD. n = 3. Two-sample Student's t-test, (**) P < 0.01. (G) Western blot analysis of DPF3 and Actin (loading control) expression in control and EBF gRNA-expressing brown adipocytes.

Fig 4: EBF2-expression drives a brown fat/thermogenic profile in primary adipocytes. (A–C) Relative mRNA levels from control or retroviral EBF2-expressing primary ingWAT stromal vascular cells with or without rosi treatment as measured by RT-qPCR. Levels of (A) Ebf2, (B) the general adipose genes Ppar?, AdipoQ and Fabp4, and (C) the brown selective genes Ucp1, Cidea, Pgc1a, Ppara and Prdm16, and (D) the mitochondrial genes Cox7a1, Cox8b, Cycs. (E) Western blot to measure protein levels of EBF2 and UCP1 levels in control or retroviral EBF2-expressing primary ingWAT stromal vascular cells following rosi treatment. P-values are represented with asterisks (*, p-value = .05; **, p-value = .01; ***, p-value = .001) and error bars represent standard deviation.

Fig 5: Ectopic ingWAT expression of EBF2 induces beiging in vivo. (A) qPCR of relative Ebf2 mRNA levels from WT (white bar) and Fabp4-Ebf2 (black bar) mice in BAT, ingWAT and epiWAT (mice housed at room temperature). (n = 3). (B) Western blot to measure EBF2 protein levels in WT or Fabp4-Ebf2 (TG) mice in ingWAT, BAT and epiWAT (mice housed at room temperature). (C) H&E staining of ingWAT from WT and Fabp4-Ebf2 mice. (D) Confocal immunofluorescence imaging of UCP1 protein levels in ingWAT from WT or Fabp4-Ebf2 mice. (E–G)) Relative mRNA levels from WT and Fabp4-Ebf2 ingWAT as measured by RT-qPCR. Levels of (E) the general adipose genes Ppar?, AdipoQ and Fabp4, (F) the brown selective genes Cidea, Pgc1a, Ppara and Prdm16 and (G) mitochondrial genes Cox7a1, Cox8b, Cycs. (H) Heat map of the top up-regulated and down-regulated genes in Fabp4-Ebf2 ingWAT compared to WT, with corresponding GO analysis. (I) Cross comparison between RNAseq data comparing WT and Fabp4-Ebf2 ingWAT gene expression and microarray comparing thermoneutral and cold exposed ingWAT. The majority of overlapping upregulated genes are related to mitochondrial function. (J) Relative UCP1 mRNA levels of WT (white bars) and Fabp4-Ebf2 (black bars) ingWAT from mice housed at thermoneutrality (TN, 30 °C) or room temperature (RT) as measured by RT-qPCR. (K) Relative oxygen consumption of ingWAT from WT and TG Fabp4-Ebf2 mice housed at room temperature, n = 4. (L) Body weight of wild type (square) and TG Fabp4-Ebf2 (circle) for mice housed at thermoneutrality on HFD beginning at 5 weeks of age. (n = 5). (M) Fat mass and lean weight mass of WT or TG Fabp4-Ebf2 (n = 5) mice after 25 weeks on HFD at TN. (N) Gross histology of ingWAT fat pads from WT or TG Fabp4-Ebf2 mice after 25 weeks on HFD at TN. P-values are represented with asterisks (*, p-value = .05; **, p-value = .01; ***, p-value = .001) and error bars denote SEM. Scale bars = 10 microns.