ebf2 whole-body knockout animals  (Johns Hopkins HealthCare)

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: EBF2 binds and regulates the chromatin state at brown fat-specific genes. ( A ) ChIP-seq profiles in reads per million total reads (RPM) for EBF2 (dark blue), H3K27ac (red), RNA polymerase II (Pol II; light blue), and PPARγ (gray) in Ebf2 wild-type and knockout BAT at Ucp1 and Ppar α. ( B ) Box plot showing changes in Pol II levels within the gene body and H3K27ac levels within 100 kb of BAT-selective (BAT-sel.), common, and WAT-selective (WAT-sel.) genes in Ebf2 knockout/wild-type BAT. Wilcoxon rank sum test, (***) P < 10 −16 . ( C ) Correlation analysis between EBF2 occupancy and differentially regulated H3K27ac peaks in Ebf2 knockout relative to wild-type BAT. ( D ) Scatter plot analysis of differentially expressed genes in Ebf2 wild-type versus knockout BAT. Fold change >1.5; false discovery rate <0.01. ( E ) Gene ontology analysis of down-regulated genes in Ebf2 knockout relative to wild-type BAT with at least one proximal EBF2-binding site within a 50-kb window around the transcription start site.

Article Snippet: Ebf2 whole-body knockout animals were obtained from Randall Reed (Johns Hopkins University) and have been described previously ( ).

Techniques: ChIP-sequencing, Knock-Out, Binding Assay

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: EBF2 interacts with the BAF complex, which incorporates the subunit DPF3 in brown adipocytes. ( A ) Coimmunoprecipitation experiment in 293T cells transfected with pcDNA3.1-Flag-EBF2 ± pMX-BRG1 followed by Flag immunoprecipitation. ( B ) Endogenous coimmunoprecipitation in differentiated mature brown adipocytes. Sheep IgG was used as a negative control. ( C ) RT-qPCR analysis of BAF subunit expression in BAT and WAT from 6-wk-old male mice. Mean ± SE. n = 3. Two-sample Student's t -test, (*) P < 0.05; (**) P < 0.01. ( D ) RT-qPCR analysis of Dpf and Ucp1 expression in inguinal white adipose following 2 wk of cold exposure at 4°C. Mean ± SE. n = 5. Two-sample Student's t -test, (**) P < 0.01. ( E ) Western blot analysis of DPF3, UCP1, and Tubulin (loading control) in inguinal WAT (iWAT) and BAT.

Article Snippet: Ebf2 whole-body knockout animals were obtained from Randall Reed (Johns Hopkins University) and have been described previously ( ).

Techniques: Transfection, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: DPF3 is required for activation of the brown fat program and mitochondrial function. ( A ) RT-qPCR analysis of day 7 mature brown adipocytes following shRNA-mediated Dpf3 depletion. ( B ) Oil-Red-O staining of shScr (control) and shDpf3 brown adipocytes. ( C ) RT-qPCR analysis of pan-adipogenic gene expression in control and DPF3-depleted mature brown adipocytes. ( D ) RT-qPCR analysis of brown fat-specific gene expression in control and DPF3-depleted mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. Mean ± SD. n = 3. Two-way ANOVA with Holm-Šídák multiple tests correction comparing shScr versus shDpf3 under basal or iso-stimlated conditions, (*) P < 0.05; (**) P < 0.01. ( E ) Western blot analysis of DPF3, EBF2, UCP1, and Actin (loading control) in control and DPF3-depleted brown adipocytes. ( F ) Oxygen consumption rate (OCR) in control and DPF3-depleted brown adipocytes; OCR was normalized to protein concentration. Mean ± SE. n = 23. Two-sample Student's t -test with Holm-Šídák multiple tests correction, (***) P < 0.001. ( G ) Uncoupled respiration in control and DPF3-depleted cells; OCR was normalized to protein concentration. Mean ± SE. n = 23. Two-sample Student's t -test, (***) P < 0.001 ( H ) OCR after acute iso stimulation in control and DPF3-depleted cells; OCR was normalized to protein concentration. Mean ± SE. n = 23. Two-sample Student's t -test, (***) P < 0.001. ( I ) Western blot analysis of DPF3, MTCO1, and Actin (loading control) in control and DPF3-depleted brown adipocytes. ( J ) Quantification of complex IV activity in control and DPF3-depleted brown adipocytes. Mean ± SE. n = 6. Two-sample Student's t -test, (***) P < 0.001. ( K ) Mean TMRE fluorescence assessed by flow cytometry in control and DPF3-depleted brown adipocytes. n = 3; 200,000 events recorded per sample. One-way ANOVA with Holm-Šídák correction for multiple comparisons, (**) P < 0.01.

Article Snippet: Ebf2 whole-body knockout animals were obtained from Randall Reed (Johns Hopkins University) and have been described previously ( ).

Techniques: Activation Assay, Quantitative RT-PCR, shRNA, Staining, Control, Gene Expression, Western Blot, Protein Concentration, Activity Assay, Fluorescence, Flow Cytometry

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: 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 β-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.

Article Snippet: Ebf2 whole-body knockout animals were obtained from Randall Reed (Johns Hopkins University) and have been described previously ( ).

Techniques: Control, ChIP-qPCR, Binding Assay, Negative Control

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: EBF2 transcriptionally regulates Dpf3 expression. ( A ) RT-qPCR analysis of Ebf2 −/− brown adipocytes following retroviral-mediated control (Puro) or EBF2 overexpression. ( B ) RT-qPCR analysis in primary inguinal adipocytes following retroviral-mediated control (Puro) or EBF2 overexpression with and without rosiglitazone included throughout differentiation. Mean ± SD. n = 3. To compare all groups in a pairwise fashion, data were analyzed using a two-way ANOVA with Holm-Šídák multiple tests correction, (*) P < 0.05; (**) P < 0.01. ( C ) RT-qPCR analysis following CRISPR-mediated gene knockout in control ( Rosa26 ) and Ebf2 knockout mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. 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. ( D ) RT-qPCR analysis of Dpf3 expression in control and Ebf2 knockout mature brown adipocytes basally or following 3 h of stimulation with 1 µM iso. Mean ± SD. n = 3. Two-way ANOVA with Holm-Šídák multiple tests correction comparing shScr versus shDpf3 under basal or iso-stimulated conditions, (**) P < 0.01. ( E ) Western blot analysis for EBF2, DPF3, and Actin (loading control) in control and Ebf2 knockout brown adipocytes. ( F ) Western blot analysis for EBF2, DPF3, UCP1, and Tubulin (loading control) in wild-type ( Ebf2 fl/fl ) or knockout ( Myf5 Cre /+ ; Ebf2 fl/fl ) BAT and iWAT. ( G ) RT-qPCR analysis of the mature adipocyte fraction from wild-type ( Ebf2 fl/fl ) or knockout ( Myf5 Cre /+ ; Ebf2 fl/fl ) animals. n = 3 animals pooled per genotype. Error bars show SD of technical replicates. Two-sample Student's t -test, (*) P < 0.05, (**) P < 0.01.

Article Snippet: Ebf2 whole-body knockout animals were obtained from Randall Reed (Johns Hopkins University) and have been described previously ( ).

Techniques: Expressing, Quantitative RT-PCR, Retroviral, Control, Over Expression, CRISPR, Gene Knockout, Knock-Out, Western Blot

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: 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 PPARγ (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.

Article Snippet: Ebf2 whole-body knockout animals were obtained from Randall Reed (Johns Hopkins University) and have been described previously ( ).

Techniques: Expressing, ChIP-sequencing, Knock-Out, ChIP-qPCR, Binding Assay, Negative Control, Quantitative RT-PCR, CRISPR, Control, Gene Expression, Western Blot

Journal: Genes & Development

Article Title: EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex

doi: 10.1101/gad.294405.116

Figure Lengend Snippet: Critical role for the histone-binding activity of DPF3 in brown adipocytes. ( A ) Schematic of the DPF3A and DPF3 domain structures. (NLS) Nuclear localization sequence; (NID) nuclear receptor interaction domain. ( B ) Flag immunoprecipitation of ectopically expressed control vector (Puro), DPF3A, or DPF3B followed by blotting for endogenous BRG1 in mature brown adipocytes. ( C ) Gene expression analysis in control (Puro), DPFA-expressing, and DPF3B-expressing mature brown adipocytes. Mean ± SD. n = 3. Two-sample Student's t -test, (*) P < 0.05; (**) P < 0.01. ( D ) Western blot analysis of control or EBF2-expressing C3H-10T1/2 cells infected with control (Puro), DPF3A, or DPF3B. ( E , F ) Gene expression analysis of common adipogenic genes ( E ) or Ucp1 ( F ) in C3H-10T1/2 cells. Mean ± SD. n = 3. Two-sample Student's t -test, (**) P < 0.01. ( G ) Model for EBF2-mediated regulation at brown fat genes.

Article Snippet: Ebf2 whole-body knockout animals were obtained from Randall Reed (Johns Hopkins University) and have been described previously ( ).

Techniques: Binding Assay, Activity Assay, Sequencing, Immunoprecipitation, Control, Plasmid Preparation, Gene Expression, Expressing, Western Blot, Infection

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: Ebf2 is required for in vivo beiging of ingWAT. (A) Confocal immunofluorescence imaging of whole-mount ingWAT tissue. EBF2 protein is present in wild type (WT) but not Ebf2 KO ingWAT. (B) H&E staining of ingWAT from WT and Ebf2 KO mice before (left panels) or after (right panels) injection with CL-316,243 to induce beiging. (C) Confocal immunofluorescence imaging of whole-mount ingWAT tissue. UCP1 protein is highly expressed in WT but not Ebf2 KO IngWAT following CL 316,243 injection. (D – G) Relative mRNA levels from in WT or Ebf2 knockout ingWAT before and after CL 316,243 injection as measured by RT-qPCR. Levels of (D) Ebf2 , (E) the general adipose genes Pparϒ, AdipoQ and Fabp4 , (F) the brown selective genes Ucp1, Cidea, Pgc1α, Pparα and Prdm16 , and (G) the mitochondrial genes Cox7a1, Cox8b, Cycs . (n = 3). (H) Relative oxygen consumption of ingWAT from WT and Ebf2 KO mice. (n = 4). P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent SEM. Scale bars = 10 microns.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: In Vivo, Immunofluorescence, Imaging, Staining, Injection, Knock-Out, Quantitative RT-PCR

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: Ebf2 is required for beiging. (A) Relative EBF2 protein levels from WT primary ingWAT stromal vascular cells plus or minus rosi treatment as assayed by western blot. (B) Relative Ebf2 mRNA levels from WT or Ebf2 KO primary ingWAT stromal vascular cells before and after rosi treatment as measured by RT-qPCR. ( C) Oil Red O staining of differentiated and rosi treated WT or Ebf2 KO primary ingWAT stromal vascular cells. (D – G) Relative mRNA levels from WT or Ebf2 KO primary ingWAT stromal vascular cells plus or minus rosi treatment as measured by RT-qPCR. Levels of (D) the general adipose genes Pparϒ and Fabp4 , (E) the brown selective genes Ucp1 and Cidea, (F) the brown selective genes Pgc1α, Pparα and Prdm16 , and (G) the mitochondrial genes Cox5b, Cox7a1, Cox8b . (H) Western blot to measure protein levels of UCP1 in differentiated and rosi treated WT or Ebf2 knockout primary ingWAT stromal vascular cells. NS = non-specific band loading control. (I) Relative Ucp1 and Pgc1α mRNA levels from WT or Ebf2 knockout primary ingWAT stromal vascular cells before and after Iso treatment as measured by RT-qPCR. P-values are represented with asterisks (*, p-value ≤ .05; **, p-value ≤ .01; ***, p-value ≤ .001) and error bars represent standard deviation.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: Western Blot, Quantitative RT-PCR, Staining, Knock-Out, Control, Standard Deviation

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: 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 Pparϒ, AdipoQ and Fabp4 , and (C) the brown selective genes Ucp1, Cidea, Pgc1α, Pparα 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.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: Expressing, Control, Retroviral, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Molecular Metabolism

Article Title: EBF2 promotes the recruitment of beige adipocytes in white adipose tissue

doi: 10.1016/j.molmet.2015.11.001

Figure Lengend Snippet: 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 Pparϒ, AdipoQ and Fabp4 , (F) the brown selective genes Cidea, Pgc1α, Pparα 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.

Article Snippet: Ebf2 whole body knockout animals were obtained from R. Reed (Johns Hopkins, Baltimore, MD, USA) and have been described previously .

Techniques: Expressing, In Vivo, Western Blot, Staining, Immunofluorescence, Imaging, Quantitative RT-PCR, Comparison, Gene Expression, Microarray