Characterization of the promoter of human adipocyte hormone-sensitive lipase.
Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The human HSL gene is composed of nine exons encoding 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 mapping experiments. Two 5'-UTRs of 70 and 170 nt respectively were obtained by rapid amplification of cDNA ends and cDNA library screenings. RNase 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 nt upstream of the start codon. In HT29, a human cell line expressing HSL, 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 respectively 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 luciferase activity was found for constructs containing the sequence between nt -2400 and -86, but not for shorter constructs. An analysis of 14 kb of genomic sequence revealed the presence of five DNase I hypersensitive sites associated with active gene transcription. Three of the sites are located in the vicinity of the transcriptional start site and could be linked to the minimal promoter activity. Two of the sites are located downstream of the exon containing the start codon, suggesting the presence of intronic regulatory elements.
Grober
J
J
Unité INSERM 317, Institut Louis Bugnard, Faculté de Médecine, Université Paul Sabatier, Bâtiment L3, CHU Rangueil, F-31403 Toulouse Cedex 4, France.
Laurell
H
H
Blaise
R
R
Fabry
B
B
Schaak
S
S
Holm
C
C
Langin
D
D
D017667:Adipocytes / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons; D015870:Gene Expression; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D002787:Sterol Esterase / Q000235:genetics*; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D002787:Sterol Esterase