The long-term objective of this study is to understand how tissue- specific gene expression is controlled. Previous studies in transgenic mice showed that cis-acting elements required to drive phosphoenolpyruvate carboxykinase (PEPCK) transcription in fat cells are different from those in liver cells. Specifically, a distal """"""""fat- specific"""""""" enhancer (from -1240 to -830 bp upstream of the PEPCK gene) is absolutely required for PEPCK expression in adipocytes. In contrast, this fat-specific enhancer is dispensable in hepatocytes. Recent studies show that: 1) a PPARgamma2/RXRalpha (peroxisome proliferator-activated receptor/9-cis retinoic acid receptor) heterodimer binds to and activates the fat-specific PEPCK enhancer via an element at -993 bp designated as PCK2; 2) PPARgamma2/RXRalpha also binds to a second site at -445 bp (designated as PCK1/AF1) which is part of a complex hormone response unit in liver; and 3) Linoleic acid induces PEPCK in cultured adipocytes.
Three specific aims are proposed to address two hypotheses regarding the mechanisms that control fat-specific PEPCK expression. This information will lead to a better understanding of how different tissue-specific factors regulate transcription from the same promoter of the PEPCK gene.. * Hypothesis l: The PPARgamma2/RXRalpha heterodimer is the critical fat- specific factor that drives PEPCK expression through PCK2, the element centered at -993 bp, within the fat-specific enhancer.
Aim l: To determine whether PPARgamma2 is the principal pairing partner for RXRalpha bound to PCK2 and whether this heterodimer drives fat- specific PEPCK expression. * Hypothesis 2: The PPARgamma2/RXRalpha heterodimer binds to PCK2 with a fixed polarity that provides an essential topology for it to interact with transcription factors bound to other sites.
Aim 2 : To determine the overall architecture of the fat-specific enhancer.
Aim 3 : To map the domain outside of the fat-specific enhancer which is needed for All enhancer activity. and to clone factors that interact with PPARgamma2.
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