Occupancy and Function of the −150 Sterol Regulatory Element and −65 E-Box in Nutritional Regulation of the Fatty Acid Synthase Gene in Living Animals

Abstract

Upstream regulatory factor (USF) and sterol regulatory element binding protein (SREBP) play key roles in the transcriptional regulation of the fatty acid synthase (FAS) gene by feeding and insulin. Due to the dual binding specificity of SREBP, as well as the presence of multiple consensus sites for these transcription factors in the FAS promoter, their physiologically relevant functional binding sites have been controversial. Here, in order to determine the occupancy of the putative USF and SREBP binding sites, we examined their protein-DNA interactions in living animals by using formaldehyde cross-linking and immunoprecipitation of chromatin and tested the function of these elements by employing mice transgenic for a reporter gene driven by various 5 deletions as well as site-specific mutations of the FAS promoter. We show that the ؊332 and ؊65 E-boxes are bound by USF in both fasted and refed mice, while the ؊150 SRE is bound by SREBP-1 only in refed mice. We also found that mutation of either the ؊150 SRE or the ؊65 E-box abolishes the feeding-induced activation of the FAS promoter in transgenic mice. Furthermore, in vivo occupancy of the FAS promoter by SREBP in the fed state can be prevented by mutation not only of the ؊150 SRE but, unexpectedly, of the ؊65 E-box as well. We conclude that the FAS promoter is activated during refeeding via the induced binding of SREBP to the ؊150 SRE and that USF binding to the ؊65 E-box is also required for SREBP binding and activation of the FAS promoter.

Sterol Regulatory Element-Binding Protein-1 Mediates the Effect of Insulin on Hexokinase II Gene Expression in Human Muscle Cells

“…This likely reflected limitations in the quantitative aspect of the ChIP method. Indeed, clear differences in the level of the interaction are generally observed in hit-andmiss situations (24,25). Under our experimental conditions, SREBP-1c was already present in the nucleus of the muscle cells (Fig.…”

NFATc4 and ATF3 Negatively Regulate Adiponectin Gene Expression in 3T3-L1 Adipocytes

“…ChIP. The ChIP assays were performed as described previously (33). Fully differentiated 3T3-L1 cells were stimulated with the indicated compounds or left unstimulated.…”

Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues

“…This physiological role of SREBP-1 in response to nutrition was initially proposed for the control of lipogenic genes such as those coding for FAS and acetyl coenzyme-A carboxylase (ACC) in the liver and adipose tissue (1). Using in vivo chromatin immunoprecipitation (ChIP) assay, direct evidence for the involvement of SREBP-1 was recently provided through the demonstration of a diet-related modulation of its binding to the promoter region of FAS and ACC-2 genes in rodent liver (19)(20)(21). In addition to lipogenic tissues, SREBP-1 mRNAs and proteins are expressed in skeletal muscle (14,22), and recent work supports its role in the regulation of glucose metabolism-related genes, such as hexokinase-2 (HKII) (10,11).…”