Figure 1. Zfp516 is a Brown Fat- Enriched Transcription Factor that binds and activates UCP1 promoter.

A. Top, diagram of Zfp516 structure. Bottom, immunoblotting for Zfp516, lamin (nuclear), and GAPDH (cytosolic) in nuclear and cytosolic fractions of HIB-1B cells. B. Top, GFP fluorescence of 293FT cells transfected with −5.5kb UCP1-GFP and either empty vector (EV) or Zfp516. Bottom, relative luciferase activity of 293FT cells cotransfected with −5.5kb UCP1-Luc and the indicated expression vector. C. Top and inlay, RT-qPCR and immunoblotting for Zfp516 mRNA and protein levels in BAT and WAT tissues from 6 week-old C57BL/6 mice (n=4 mice). Bottom, RT-qPCR for Zfp516 and UCP1 mRNA levels in the adipocyte fraction and SVF from BAT. D. Schematic representation of 5’ deletion constructs of the UCP1 promoter-luciferase and relative luciferase activity in 293FT cells cotransfected with indicated promoter construct and Zfp516 (expressed as fold of vector control). E. Top, ChIP for Zfp516 association to the UCP1 promoter in BAT. Bottom, qPCR for the ChIP DNA for Zfp516 association to the UCP1 promoter in BAT. F. Top-left, immunoblotting of lysates from 293FT cells transfected with −5.5kb UCP1-GFP with Flag-Zfp516 or vector. Top-right, ChIP for Flag-Zfp516 association to the UCP1 promoter using both αFlag and αZfp516 antibodies. Bottom, qPCR quantification of ChIP DNA for the association of full length Zfp516 or truncations of Zfp516 to the UCP1 promoter in 293FT cells. See also Figure S1, S2, and S3. *p<0.05; **p<0.01; ***p<0.001