Abstract
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 -200 to -126. 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.
Highlights
We have previously shown that the proximal 2-kb se- positive regulators, whereas cAMP [1] and polyunsaturated quence of the fatty acid synthase (FAS) promoter trans- fatty acids [4] are able to suppress FAS gene transcription in an fected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese versus lean Zucker rat adipocytes
Deletional Analysis of FAS Promoter Activity in Primary Cultured Adipocytes: Evidence of a Strong Negative Regulatory Region between Ϫ200 and Ϫ126 —We have previously shown that the 5Ј-flanking 2-kb region of the rat FAS gene is able to direct a high level of reporter gene activity in transfected rat adipocytes through correctly initiated transcription [11]
The results show that the Sp1 mutation elicited a 2.5-fold increase in CAT expression in adipocytes from lean rats, suggesting that the Sp1 binding site is partly responsible for the negative effect of the Ϫ200 to Ϫ126 region on FAS gene transcription
Summary
We have previously shown that the proximal 2-kb se- positive regulators, whereas cAMP [1] and polyunsaturated quence of the fatty acid synthase (FAS) promoter trans- fatty acids [4] are able to suppress FAS gene transcription in an fected into rat adipocytes was highly sensitive to the cellular context, displaying an overactivity in obese (fa/ fa) versus lean Zucker rat adipocytes. 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 the 2-kb promoter region that play a role in FAS gene transcription in the context of the mature unilocular rat adipocyte, and 2) to identify the region(s) involved in the responsiveness of FAS promoter to adipocyte fa-dependent transcription factors. We highlighted a 74-bp (Ϫ200 to Ϫ126) functional motif as the critically important control region for defining genotype-specific differences in the level of FAS gene transcription This motif was found to exert a strong negative control on FAS promoter activity in lean but not in obese rat adipocytes. Insulin [1], thyroid hormone [2], and glucose [3] act as
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