Human fatty-acid synthase gene. Evidence for the presence of two promoters and their functional interaction.
We have isolated and sequenced a genomic clone coding for the first three exons and the 5'-flanking region of the human fatty-acid synthase gene. The translation initiation site, ATG, is located in exon II. Primer extension and S1 nuclease analyses showed the presence of three transcription initiation (Ti) sites: Ti I, Ti II, and Ti III. The Ti I site is mapped to the beginning of the untranslated exon I and preceded by a promoter with recognizable TATA and CAAT boxes. The Ti II and Ti III sites are located in intron I, at 60 and 49 nucleotides upstream of the translation initiation site ATG in exon II, respectively. These two Ti sites are preceded by four putative Sp1 boxes, but lack TATA and CAAT boxes. Analysis of luciferase reporter gene expression in transient transfection assays confirmed the existence of two promoters. A 200-base pair 5'-flanking region, which has strong promoter activity comparable with that of the CMV promoter, is considered human fatty-acid synthase promoter I. In a wild-type human fatty-acid synthase-luciferase construct, in which promoter I and intron I are present in their natural configuration, the reporter gene activity is only 1% of that of promoter I. Deletion analysis showed the existence of promoter II, which is located in intron I immediately upstream of the Ti II site. The strength of promoter II is approximately th of that of promoter I in transient transfection assays. Further analysis of reporter gene constructs showed that promoter II inhibited the reporter gene activity of the wild-type construct that contained promoter I and intron I and that the spatial separation of the two promoters is important for this inhibition. A model is proposed based on the possibility that the assembly of transcription complexes on promoter II creates a "roadblock" and reduces the overall expression of the fatty-acid synthase gene by interfering with the progression of transcription from promoter I.
Hsu
M H
MH
Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Chirala
S S
SS
Wakil
S J
SJ
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D064429:Fatty Acid Synthases / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D017384:Sequence Deletion; D016385:TATA Box; D014158:Transcription, Genetic
D012333:RNA, Messenger; D004247:DNA; D008156:Luciferases; D064429:Fatty Acid Synthases