A GC-rich Region Containing Sp1 and Sp1-like Binding Sites Is a Crucial Regulatory Motif for Fatty Acid Synthase Gene Promoter Activity in Adipocytes

We have previously shown that the proximal 2-kb sequence of the fatty acid synthase (FAS) promoter transfected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese (fa/ fa) versus lean Zucker rat adipocytes. Using deletional analysis, we show here that FAS promoter activity mainly depends on a region from 2200 to 2126. This sequence exerts a strong negative effect on FAS promoter in adipocytes from lean rats but not in those from obese rats, resulting in a marked overtranscriptional activity in the latter cells. This region, fused to a heterologous promoter, the E1b TATA box, induced differential levels of gene reporter activity in lean and obese rat adipocytes, indicating it harbors fa-responsive element(s). Whatever the rat genotype, adipocyte nuclear proteins were shown to footprint the same protected sequence within the fa-responsive region, and supershift analysis demonstrated that Sp1 or Sp1-like proteins were bound to this DNA subregion. Compelling evidence that the Sp1 binding site contained in this sequence was implicated in the differential promoter activity in lean versus obese rats, was provided by the observation that mutations at this Sp1 site induced a 2.5-fold increase in FAS promoter activity in adipocytes from lean rats, whereas they had no effect in adipocytes from obese rats. Fatty acid synthase (FAS) 1 is a multifunctional enzyme that catalyzes all the reaction steps in the conversion of acetyl-CoA and malonyl-CoA to palmitate. FAS plays a central role in de novo lipogenesis, and its level of expression is a key determinant of the maximum capacity of a tissue to synthesize fatty acids. In mammals, FAS is expressed at especially high levels in liver and adipose tissue, where it greatly contributes to the regulation of triglyceride-rich lipoprotein production and to appropriate fat storage. In these tissues, FAS enzyme concentrations are under strict nutritional and hormonal control, and many studies using animal models or established cell lines have determined that the regulation of FAS activity is exerted mainly at the transcriptional level of gene expression. In particular, insulin (1), thyroid hormone (2), and glucose (3) act as positive regulators, whereas cAMP (1) and polyunsaturated