Early B Cell Factor Activity Controls Developmental and Adaptive Thermogenic Gene Programming in Adipocytes

Abstract

Brown adipose tissue (BAT) activity protects animals against hypothermia and represents a potential therapeutic target to combat obesity. The transcription factor early B cell factor-2 (EBF2) promotes brown adipocyte differentiation, but its roles in maintaining brown adipocyte fate and in stimulating BAT recruitment during cold exposure were unknown. We find that the deletion of Ebf2 in adipocytes of mice ablates BAT character and function, resulting in cold intolerance. Unexpectedly, prolonged exposure to cold restores the thermogenic profile and function of Ebf2 mutant BAT. Enhancer profiling and genetic assays identified EBF1 as a candidate regulator of the cold response in BAT. Adipocyte-specific deletion of both Ebf1 and Ebf2 abolishes BAT recruitment during chronic cold exposure. Mechanistically, EBF1 and EBF2 promote thermogenic gene transcription through increasing the expression and activity of ERRα and PGC1α. Together, these studies demonstrate that EBF proteins specify the developmental fate and control the adaptive cold response of brown adipocytes.

Keywords: EBF1; EBF2; ERR; PGC1; UCP1; brown adipocyte; brown fat; cold exposure; thermogenesis.

Figure 1.. Adipocyte EBF2 Controls BAT Thermogenic Gene Expression and Activity

(A) Ebf2 mRNA levels in the stromal-vascular fraction of BAT from 3–4-week-old Ebf2^WT, Ebf2^ΔMyf5, and Ebf2^ΔAdipoq mice (n = 3–5 mice per group; mean ± SEM). (B) Hematoxylin and eosin staining of BAT from P0.5 Ebf2^WT and Ebf2^ΔAdipoq mice (scale bar, 100 μm). (C) Western blot analysis of UCP1 and mitochondrial respiratory chain components in BAT from P0.5 Ebf2^WT and Ebf2^ΔAdipoq mice. (D) Electron micrographs of BAT from P0.5 Ebf2^WT and Ebf2^ΔAdipoq mice (scale bar, 500 nm; arrow-head: mitochondria; L: lipid droplet). (E) Relative mRNA levels of indicated genes in BAT from 8- to 10-week-old Ebf2^WT and Ebf2^ΔAdipoq mice housed at room temperature (RT; 24°C) (n = 5 mice per group; mean ± SEM). (F) Cold-tolerance test in RT-acclimated, 8- to 10-week-old Ebf2^WT and Ebf2^ΔAdipoq mice (n = 8–10 mice per group; mean ± SEM). (G) Volume of O₂ (VO₂) consumed following norepinephrine (NE) treatment of Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT (n = 7–10 mice per group; mean ± SEM).

Figure 2.. EBF2 Is Not Required for Chronic Cold-Induced Thermogenic BAT Recruitment

(A) Hematoxylin and eosin staining of BAT in Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT or 30°C (thermoneutrality [TN]) for 1 month (scale bar, 100 μm). (B) Relative mRNA levels of Ebf2, Ucp1, and Cycs in BAT from Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT or TN (n = 5–6 mice per group; mean ± SEM). (C) Western blot analysis of UCP1 and mitochondrial respiratory chain components in BAT from Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT or TN. Tubulin was used for loading control. (D) Relative mRNA levels of indicated genes in BAT from Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT or 4°C for 1 week (n = 9–11 mice per group; mean ± SEM). (E) Western blot analysis of UCP1 and mitochondrial respiratory chain components in BAT from Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT or 4°C for 1 week. (F) VO₂ consumed following NE treatment of Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT or 4°C for 1 week (n = 5–7 mice per group; mean ± SEM). (G) Relative mRNA levels of Ucp1 and Cycs in BAT from Ebf2^WT and Ebf2^ΔAdipoq mice housed at (1) RT, (2) 4°C for 1 week, or (3) 4°C for one week, then RT for 1 week (4C/RT) (n = 3–6 mice per group; mean ± SEM).

Figure 3.. EBF Activity Controls Basal and Adaptive Thermogenic Gene Expression

(A) Clustering analysis of gene expression in BAT from Ebf2^WT and Ebf2^ΔAdipoq mice housed at RT or 4°C for 1 week (n = 3 mice per group). (B) Gene Ontology analysis of genes in the light green cluster. (C) Heatmaps of selected genes from light green cluster in (A). Mean log₂ fold change (FC). (D) De novo motif analysis of regions with decreased H3K27ac levels in Ebf2 mutant versus control (WT) BAT of mice housed at RT. (E and F) De novo motif analysis of regions that display increased H3K27ac levels during cold exposure in BAT from (E) Ebf2^WT and (F) Ebf2^ΔAdipoq mice. (G) Relative mRNA levels of Ebf1, Ebf2, and other indicated genes in BAT from Ebf1^WT and Ebf1^ΔAdipoq mice housed at RT (n = 4 mice per group; mean ± SEM). (H) Hematoxylin and eosin staining of BAT from Ebf2^WT, Ebf2^ΔAdipoq, Ebf1/2^WT, and Ebf1/2^ΔAdipoq mice housed at RT or 4°C for 1 week (scale bar, 100 μm). (I) Relative mRNA levels of indicated genes in BAT from Ebf1/2^WT (control) and Ebf1/2^ΔAdipoq (double knockout [DKO]) mice housed at RT or 4°C for 1 week (n = 3–4 mice per group; mean ± SEM).

Figure 4.. EBF2 Cooperates with ERRα and PGC1α to Promote Ucp1 Transcription

(A) Chromatin immunoprecipitation sequencing (ChIP-seq) tracks of indicated transcription factors and histone modifications at the Ucp1 locus (Emmett et al., 2017; Shapira et al., 2017). (B) Schematic depicting Ucp1 −6 kb reporter construct and putative EBF and ERR binding sites deleted in mutant reporter. (C and D) Transcriptional activity of the Ucp1 −6 kb enhancer in NIH 3T3 cells upon expression of (C) EBF2, PGC1α, and/or ERRα; (D) EBF2, EBF2 R162A (DNA-binding mutant) or EBF2 DTH (C-terminal domain mutant); and/or PGC1α (n = 3 replicates per group; mean ± SEM). (E–G) Transcriptional activity of the WT or mutant (Mut.) Ucp1 −6 kb enhancer in NIH 3T3 cells upon expression of (E) ERRα and/or PGC1α (n = 3); (F) EBF2 and/or PGC1α (n = 3); or (G) EBF2 and/or PPARγ/RXRα (n = 9) (mean ± SEM). (H) Transcriptional activity of the Ucp1 −6 kb enhancer in control (ctl) or Esrra-deficient NIH 3T3 cells upon expression of EBF2 and/or PGC1α (n = 3 replicates per group; mean ± SEM). (I) Model of EBF function at thermogenic genes (i.e., Ucp1) in the context of cold exposure.