. 2013 Apr 2;17(4):562-74.
Epub 2013 Mar 14.
EBF2 Determines and Maintains Brown Adipocyte Identity
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EBF2 Determines and Maintains Brown Adipocyte Identity
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The master transcription factor Pparγ regulates the general differentiation program of both brown and white adipocytes. However, it has been unclear whether Pparγ also controls fat lineage-specific characteristics. Here, we show that early B cell factor-2 (Ebf2) regulates Pparγ binding activity to determine brown versus white adipocyte identity. The Ebf DNA-binding motif was highly enriched within brown adipose-specific Pparγ binding sites that we identified by genome-wide ChIP-Seq. Of the Ebf isoforms, Ebf2 was selectively expressed in brown relative to white adipocytes and was bound at brown adipose-specific Pparγ target genes. When expressed in myoblasts or white preadipose cells, Ebf2 recruited Pparγ to its brown-selective binding sites and reprogrammed cells to a brown fat fate. Brown adipose cells and tissue from Ebf2-deficient mice displayed a loss of brown-specific characteristics and thermogenic capacity. Together, these results identify Ebf2 as a key transcriptional regulator of brown fat cell fate and function.
Copyright © 2013 Elsevier Inc. All rights reserved.
Figure 1. The Ebf motif is highly enriched at BAT-specific Pparγ binding sites
(A) ChIP for Pparγ followed by massively parallel sequencing and genome alignment was used to profile binding sites in mouse eWAT and BAT. (B) Representative Pparγ ChIP-sequencing tracks in BAT (brown) and eWAT (blue) at common, BAT-specific and WAT-specific sites. (C) Independent ChIP-qPCR validation of Pparγ binding in BAT and eWAT at select sites (mean ± SD; n=3; *p<0.05, **p<0.01). (D) Motif analyses of BAT- and eWAT-specific Pparγ binding regions.
Figure 2. Ebf2 is selectively expressed in brown relative to white adipose cells and tissue
(A) mRNA levels of
Ebf1, Ebf2 and Ebf3 in eWAT, ingWAT (inguinal) and BAT (mean ± SD; n=3; ** p <0.01). (B) mRNA levels of Ebf1, Ebf2 and Ebf3 in a panel of white and brown adipose cell lines (mean ± SD; n=3; ** p <0.01). (C) Western blot analysis of Ebf1, Ebf2 and Ebf3 protein expression in representative samples from eWAT, BAT (left panel) and adipocytes (day 7) from white (3T3-L1) and brown (BAT 1) preadipocytes. Tbp was used for a loading control. (D) ChIP-qPCR analysis of Ebf2 binding in BAT at sites bound by Pparγ in: BAT and WAT (common), BAT-only and WAT-only (mean ± SD; n=3–5).
Figure 3. Ebf2 expression drives a brown fat-specific differentiation program
(A) Ctl- and Ebf2-expressing C2C12 myoblasts were induced to differentiate into adipocytes followed by staining of triglycerides with oil-red-o. (B, C) Above cultures were analyzed for their expression levels of:
Pparγ and AdipoQ (B); and Ucp1 levels ± Isoproterenol treatment (C) (n=3). (D–I) Ctl- and Ebf2-expressing white preadipocytes were induced to differentiate into adipocytes followed by staining with oil-red-o (D). (E–H) mRNA levels of: Ebf2 and general adipocyte markers (E); brown-selective genes (F); mitochondrial genes (G); Ucp1 and Pgc1α ± Isoproterenol treatment (H) (n=3–5 pools/construct). (I) Bodipy staining of triglycerides (green) and Ucp1 immunostaining (red). All expression data are: mean ± SD; * p <0.05; ** p <0.01.
Figure 4. Ebf2 is required to maintain the expression of brown-specific genes in brown adipocytes
(A) Phase contrast micrographs of brown adipocyte cultures 2 days after treatment with an Ebf2-specific siRNA or a siSCR control. (B–D) mRNA levels of:
Ebf2 (B); general fat markers (C); brown-specific and mitochondrial genes (D). (E) Western blot analysis of Ebf2, Pparγ, Ucp1 and Tubulin (loading control) protein levels. Expression data are mean ± SD; n=3; **p<0.01.
Figure 5. Ebf2 and Pparγ cooperatively activate transcription
(A) Pparγ ChIP-seq tracks from BAT (brown) and WAT (blue) at the
Prdm16 locus. (B) ChIP-qPCR analysis of Ebf2 and Pparγ binding to the −25 kb Prdm16 region (red box in (A)) in eWAT and BAT. (C) Transcriptional activity of the −25 kb region of Prdm16 or control reporter construct (pGL4.24) in response to expression of: Ebf2, Pparγ/Rxrα, or the combination of Ebf2 and Pparγ/Rxrα in NIH-3T3 cells (n=4; mean ± SD). (D) Western blot analysis of Ebf2 and Pparγ levels in NIH-3T3 lysates that were used for transcription assays in (C). (E) ChIP-qPCR analysis of Ebf2 and Pparγ binding to the −25 kb Prdm16 region and Fabp4 during a time-course of brown adipocyte differentiation.
Figure 6. Ebf2 promotes the binding of Pparγ to its brown fat-selective gene targets
(A) ChIP-qPCR analysis of Pparγ binding in adipocytes from Pparγ-deficient cells that were first transduced with Pparγ retrovirus, followed by transduction with either a control (ctl) or Ebf2-expressing retrovirus. (B) ChIP-qPCR analysis of Pparγ binding in Scrambled (Ctl) or Si-Ebf2 treated brown adipocytes. (C) ChIP-qPCR analysis of Pparγ binding in mature adipocytes from control (Ctl) and Ebf2-expressing white preadipocytes. All data are normalized to background binding (at non-specific
Insulin and 18s rRNA loci) and presented as mean ± SD, n > 3.
Figure 7. Ebf2 is required for BAT development in mice
(A) Hematoxylin and Eosin (H&E) staining of representative sections from the interscapular region of wildtype and
Ebf2-deficient embryos at E18 dpc. (B, C) BAT from Ebf2 and +/− Ebf2 embryos (E18) was examined by real-time PCR analysis for mRNA levels of: general adipocyte genes, brown-specific genes and mitochondrial genes (B); and WAT-selective genes (C) (n=5–8 embryos/genotype). (D) Brown fat precursors from −/− Ebf2, +/+ Ebf2 and +/− Ebf2 embryos were differentiated into adipocytes in culture and stained with oil-red-o. (E–H) Above cultures were analyzed by real-time qPCR for their expression levels of: Ebfs (E); general adipocyte markers (F); brown-specific genes (G); and white-selective genes (H) (n=3 pools [> 5 mice per pool]). (I) Oxygen consumption in cultured brown adipocytes from −/− Ebf2 and +/− Ebf2 before and after stimulation with norepinephrine (NE) (mean ± SD; n=3). Expression data are: mean ± SD, * p <0.05; ** p <0.01. −/−
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Adipocytes, Brown / cytology
Adipocytes, Brown / metabolism*
Basic Helix-Loop-Helix Transcription Factors / deficiency
Basic Helix-Loop-Helix Transcription Factors / genetics
Basic Helix-Loop-Helix Transcription Factors / metabolism*
DNA-Binding Proteins / genetics
DNA-Binding Proteins / metabolism
High-Throughput Nucleotide Sequencing
Protein Isoforms / metabolism
Transcription Factors / genetics
Transcription Factors / metabolism
Basic Helix-Loop-Helix Transcription Factors
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