Two 5′-Regions Are Required for Nutritional and Insulin Regulation of the Fatty-acid Synthase Promoter in Transgenic Mice

• Published in: The Journal of biological chemistry Vol. 275; no. 14; pp. 10121 - 10127
• Main Authors: Moon, Yang Soo, Latasa, Maria-Jesus, Kim, Kee-Hong, Wang, Dong, Sul, Hei Sook
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.04.2000
• Subjects: 5' Untranslated Regions; 5' Untranslated Regions - genetics; adipocytes; adipose tissue; Adipose Tissue - enzymology; Animals; biosynthesis; chloramphenicol acetyltransferase; Chloramphenicol O-Acetyltransferase; Chloramphenicol O-Acetyltransferase - genetics; diet; drugs; enzymology; FAS gene; Fatty Acid Synthases; Fatty Acid Synthases - biosynthesis; Fatty Acid Synthases - genetics; fatty-acid synthase; gene expression; Gene Expression Regulation, Enzymologic; genetic regulation; genetics; high carbohydrate diet; hormonal regulation; insulin; liver; Liver - enzymology; luciferase; messenger RNA; Mice; Mice, Transgenic; promoter regions; Promoter Regions, Genetic; Rats; recombinant DNA; Recombinant Fusion Proteins; Recombinant Fusion Proteins - biosynthesis; repletion; reporter genes; Restriction Mapping; RNA, Messenger; RNA, Messenger - genetics; starvation; streptozotocin; transcription (genetics); transcription factors; transgenic animals
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.14.10121

We previously reported that 2.1 kilobase pairs of the 5′-flanking sequence are sufficient for tissue-specific and hormonal/metabolic regulation of the fatty-acid synthase (FAS) gene in transgenic mice. We also demonstrated that the −65 E-box is required for insulin regulation of the FAS promoter using 3T3-L1 adipocytes in culture. To further define sequences required for FAS gene expression, we generated transgenic mice carrying from −644, −444, −278, and −131 to +67 base pairs of the rat FAS 5′-flanking sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Similar to the expression observed with −2100-FAS-CAT transgenic mice, transgenic mice harboring −644-FAS-CAT and −444-FAS-CAT expressed high levels of CAT mRNA only in lipogenic tissues (liver and adipose tissue) in a manner identical to the endogenous FAS mRNA. In contrast, −278-FAS-CAT and −131-FAS-CAT transgenic mice did not show appreciable CAT expression in any of the tissues examined. When previously fasted mice were refed a high carbohydrate, fat-free diet, CAT mRNA expression in transgenic mice harboring −644-FAS-CAT and −444-FAS-CAT was induced dramatically in liver and adipose tissue. The induction was virtually identical to that observed in −2100-FAS-CAT transgenic mice and to the endogenous FAS mRNA. In contrast, −278-FAS-CAT transgenic mice showed induction by feeding, but at a much lower magnitude in both liver and adipose tissue. The −131-FAS-CAT transgenic mice did not show any CAT expression either when fasted or refed a high carbohydrate diet. To study further the effect of insulin, we made these transgenic mice insulin-deficient by streptozotocin treatment. Insulin administration to the streptozotocin-diabetic mice increased CAT mRNA levels driven by the −644 FAS and −444 FAS promoters in liver and adipose tissue, paralleling the endogenous FAS mRNA levels. In the case of −278-FAS-CAT, the induction observed was at a much lower magnitude, and deletion to −131 base pairs did not show any increase in CAT expression by insulin. This study demonstrates that the sequence requirement for FAS gene regulation employing an in vitroculture system does not reflect the in vivo situation and that two 5′-flanking regions are required for proper nutritional and insulin regulation of the FAS gene. Cotransfection of the upstream stimulatory factor and various FAS promoter-luciferase constructs as well as in vitro binding studies suggest a function for the upstream stimulatory factor at both the −65 and −332 E-box sequences.