Fig 2: Conditional deletion of TLE3 in adipocytes protects against diet-induced weight gain and glucose intolerance. (A) Time course of body weight for control (Tle3F/F) and knockout (Tle3F/F Adipoq-Cre) mice housed at ambient temperature on a 60% HFD for 14 wk. n = 6–8 per group. (B) Body fat composition by NMR in Tle3F/F and Tle3F/F Adipoq-Cre groups on a 60% HFD over time. n = 6–8 per group. (C) GTT and AUGg on Tle3F/F and Tle3F/F Adipoq-Cre mice after 14 wk on a 60% HFD. n = 6–7 per group. (D) ITT and AUCg on Tle3F/F and Tle3F/F Adipoq-Cre mice after 10 wk on a 60% HFD. n = 3 per group. (E) Weight of individual iWAT tissue depots. n = 10 per group. (F) Histology of adipose depots after 14 wk on a 60% HFD. Data are presented as mean ± SEM. Significance was analyzed by two-tailed Student's t-test. (*) P < 0.05; (**) P < 0.01.

Fig 3: Analysis of TLE3 binding demonstrates repressive interaction with EBF2. (A) De novo motif analysis of the top 1000 TLE3 occupancy sites in WAT and their assigned closest matching transcription factor, listed in order of decreasing P-value. (B) Known motif analysis of the EBF motif in TLE3-binding sites and matching background sequences within 15 kb of the indicated gene groups. (C) Overlap between TLE3- and EBF2-binding sites within 15 kb of the indicated gene groups with randomly selected GC content-matching background sequences. (D) EBF2 ChIP-seq signal in TLE3-binding sites within 15 kb of the indicated gene groups. (E) Coimmunoprecipitation of TLE3 and EBF proteins from 293 cells transfected with Flag-TLE3 and one of the following: EBF1-V5, EBF2-V5, or EBF3-V5. Lysates were immunoprecipitated with anti-V5 antibody. (F) Coimmunoprecipitation of TLE3 and EBF2 from Tle3F/F iWAT adipocytes expressing CreERT2 and treated with either EtOH (control) or 4-hydroxytamoxifen. Lysates were immunoprecipitated with anti-TLE3 antibody. (G) Analysis of Cldn1 enhancer activation of luciferase reporter by coexpression of EBF2, TLE3, or both in beige adipocytes differentiated in vitro. n = 3 per group. (H) Gene expression of Tle3F/F iWAT adipocytes expressing CreERT2. Cells treated with DMSO (control) or 4-hydroxytamoxifen on day 4 of differentiation and either siSCR or siEBF2 on day 12 of differentiation and harvested on day 14 of differentiation. n = 3 per group. Data are presented as mean ± SEM. Significance was analyzed by Fisher's exact test (B,C), Wilcoxon rank sum test (D), or Student's t-test (G,H). (*) P < 0.05; (**) P < 0.01; (***) P < 2.2 × 10−8; (****) P < 2.2 × 10−16.

Fig 4: TLE3 represses TCA cycle and thermogenic gene program in iWAT. (A) Volcano plot showing differentially expressed genes in beige adipocytes isolated from Tle3F/F mice expressing CreERT2. Cells were treated with EtOH (control) or 4-hydroxytamoxifen on day 4 after differentiation and harvested on day 10. Adjusted P-value of <0.01; log2 ratio of differential expression >|0.2|. (B) Stacked bar graph demonstrating genes that are differentially regulated with the loss of TLE3 with or without a TLE3-binding site. RNA-seq genes with adjusted P-value of <0.05, no distance from TSS cutoff applied. (C) Gene ontology analysis of 475 genes whose expression is increased with loss of TLE3 and which have at least one TLE3 occupancy site identified by ChIP-seq. Reactome pathways (P < 0.0005) are listed in order of decreasing P-value. (D) Gene ontology analysis of 469 genes whose expression is decreased with loss of TLE3 and which have at least one TLE3 occupancy site identified by ChIP-seq. Reactome pathways (P < 0.0005) are listed in order of decreasing P-value. (E) Heat map of genes associated with Reactome pathway R-MMU-163200 (respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins) in Tle3F/F iWAT adipocytes expressing CreERT2 and treated with either EtOH (control) or 4-hydroxytamoxifen. Z-score reflects the log2 ratio. n = 3 per group.

Fig 5: Genome-wide profiling of TLE3 in adipocytes reveals increased binding in WAT. (A) Venn diagram demonstrating the number of TLE3-binding sites in eWAT, iWAT, and BAT, filtered for >15 tags and >10-fold above input. (B) Binding intensity at sites occupied by TLE3 in differentiated adipocytes from eWAT, iWAT, and BAT. (C) Genomic distribution of TLE3 relative to the distance from transcriptional start sites (TSSs). (D) Genome browser tracks of Fabp4 for TLE3 and PPAR? in eWAT, iWAT, and BAT. (E) Genome browser tracks of Ppargc1a for TLE3 and PPAR? in eWAT, iWAT, and BAT. (F) Genome browser tracks of Ucp1 for TLE3 and PPAR? in eWAT, iWAT, and BAT.