CHARACTERIZATION OF THE PROMOTER OF HUMAN ADIPOCYTE HORMONE-SENSITIVELIPASE

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adi pose tissue lipolysis. The human HSL gene is composed of nine exons en coding the adipocyte form and a testis-specific coding exon. Northern blot analyses showed that human adipocytes express a 2.8 kb HSL mRNA, suggesting the presence of a short (20-150 bp) 5' untranslated region (5'-UTR). A single 5'-UTR of approx. 70 nt was detected in RNase H map ping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtai ned by rapid amplification of cDNA ends and cDNA library screenings. R Nase protection experiments, with probes derived from the two products , showed that human adipocyte HSL mRNA contains only the 70 nt product . Primer extension analysis mapped the transcriptional start site 74 n t upstream of the start codon. In HT29, a human cell line expressing H SL, the presence of the short or the long 5'-UTR is mutually exclusive . The short and long 5'-UTR exons were located 1.5 and approx. 13 kb r espectively upstream of the first coding exon. Various portions of the 5'-flanking region upstream of the short product exon were linked to the luciferase gene and transfected into cells that express HSL (HT29 cells and rat adipocytes) and do not express HSL (HeLa cells). High lu ciferase activity was found for constructs containing the sequence bet ween nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypers ensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site a nd could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggest ing the presence of intronic regulatory elements.