Identification and characterization of the human adipocyte apM-1 promoter

Abstract

The human adipocyte-specific apM-1 gene encodes a secretory protein of the adipose tissue and seems to play a role in the pathogenesis of obesity. A 1.3 kb amount of the proximal promoter region has been cloned and analyzed for the presence of putative transcription factor binding sites. Several binding sites known to be involved in adipogenesis and regulation of adipocyte-specific genes (C/EBP, SREBP) are present. No TATA box, but a classical CCAAT box could be identified. To confirm functionality and cell specificity of the 1.3 kb promoter, a series of 5'-deleted fragments were ligated in front of the luciferase gene and the constructs were transfected into 3T3-L1 adipocytes. The reporter gene was effectively transcribed, as demonstrated by the expression of enzyme activity. The 5'-end of the human cDNA was completed by 5'-RACE-PCR. Several alternative transcription start sites were detected by RNase protection assay and primer extension analysis. In addition, an exon/intron boundary was mapped at the extreme 5'-end of the cDNA sequence. Genomic Southern blotting suggests that the human apM-1 gene is a single copy gene.