Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.
Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition 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 in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-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 protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.
Hwang
C S
CS
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Mandrup
S
S
MacDougald
O A
OA
Geiman
D E
DE
Lane
M D
MD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D020738:Leptin; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D020738:Leptin; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D008156:Luciferases