TRANSCRIPTIONAL ACTIVATION OF THE MOUSE OBESE (OB) GENE BY CCAAT ENHANCER-BINDING PROTEIN-ALPHA

Like other adipocyte genes that are transcriptionally activated by CCA AT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte di fferentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking regio n of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage in hibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level i n preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobili ty-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific prot ein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP bind ing sites failed to generate protein-oligonucleotide complexes. Cotran sfection of a C/EBP alpha expression vector into 3T3-L1 cells with a s eries of 5' truncated ob gene promoter constructs activated reporter g ene expression with all constructs containing the proximal C/EBP bindi ng site (nucleotides -55 to -47). Mutation of this site blocked transa ctivation by C/EBP alpha. Taken together, these findings implicate C/E BP alpha as a transcriptional activator of the ob gene promoter and id entify the functional C/EBP binding site in the promoter.