(A) Silver staining shows co-immunoprecipitated proteins in FLAG-V5-GFP-expressing (GFP, lane 1) and FLAG-V5-TAF7L-expressing (TAF7L, lane 2) C3H10T1/2 differentiated fat cells. Comparative LC-MS/MS analysis identified peptides matching with TFIID subunits (TAF1, TAF4, TBP, TAF5, TAF6, and TAF10) and PPARγ in TAF7L-expressing but not in GFP-expressing cells. FKBP15 and ACSL1 are representative non-specific associated proteins. (B) Western blot analyzing input and immunoprecipitated protein levels of PPARγ, TAF4, TAF7L, and TBP in samples from A. (C) HA-tagged TAF7L and FLAG-tagged PRDM16 were overexpressed in 293T cells, immunoprecipitations were performed on both FLAG and HA antibodies and followed by Western blotting with FLAG and HA antibodies. (D) Upper panel, schematic picture shown the distance between distal enhancer (D) and core promoter (P) of Cidea gene; middle panel, read accumulation of TAF7L, TBP, and PPARγ on Cidea locus in differentiated fat cells from ChIP-seq analysis; bottom panel, 3C experiments assess long-range DNA interactions between the TAF7L/PPARγ binding distal enhancer (D) and core promoter (P) sites of Cidea in WT and Taf7l KO BAT. ▲, anchor point. Also see Figure 3—figure supplement 1C. (E) Model shows TAF7L-mediating regulatory DNA looping to specify BAT differentiation from mesenchymal stem cells (MSC).