Figure 5

Coup-TFII binds to a conserved element of the Ucp3 promoter. (A) Electrophoretic mobility shift assay with candidate probes A-E and nuclear extracts of HEK293 cells. Overexpression of Coup-TFII leads to formation of a specific complex on probeC only, that can be supershifted with a Coup-TFII antibody. All complexes are subject to competition with 100 fold molar excess of unlabelled probe C. (B) Western Blot of protein fractions of HEK293 cells with a Coup-TFII-antibody. Coup-TFII is specifically detected in the nuclear fraction and absent in untransfected cells. M = Marker, - = mock transfected, CI = Coup-TFI transfected, CII = Coup-TFII transfected. (C) Alignment of the human and rodent promoter regions of the Ucp3 gene (Ps: -821 to -857). The positions of identical bases are marked (*). The putatively Coup-TFII binding elements (box, dashed box) located on probe C are conserved in all rodent species. In the murine promoter they are found in reverse order slightly more upstream. The human promoter does not feature comparable sequences in the compared region. Rn = R. norvegicus, Mm = M. musculus, Ps = P. sungorus, Hs = H. sapiens (D) Electrophoretic mobility shift assay with skeletal muscle nuclear extracts from control and food deprived hamsters. The complex shown is of a size comparable to the Coup-TFII complex above. Starvation induces complex formation in comparison to control conditions.