Adipose expression of the phosphoenolpyruvate carboxykinase promoter requires peroxisome proliferator-activated receptor gamma and 9-cis-retinoic acid receptor binding to an adipocyte-specific enhancer in vivo

A putative adipocyte-specific enhancer has been mapped to approximately 1 k ilobase pair upstream of the cytosolic phosphoenolpyruvate carboxykinase (P EPCK) gene, In the present study, we used transgenic mice to identify and c haracterize the 413-base pair (bp) region between -1242 and -828 bp as a bo na fide adipocyte-specific enhancer in vivo. This enhancer functioned most efficiently in the context of the PEPCK promoter. The nuclear receptors per oxisome proliferator-activated receptor gamma (PPAR gamma) and g-cis-retino ic acid receptor (RXR) are required for enhancer function in vivo because: 1) a 3-bp mutation in the PPAR gamma-/RXR-binding element centered at -992 bp, PCK2, completely abolished transgene expression in adipose tissue; and 2) electrophoretic mobility supershift experiments with specific antibodies indicated that PPAR gamma and RXR are the only factors in adipocyte nuclea r extracts which bind PCK2, In contrast, a second PPAR gamma/RXR-binding el ement centered at -446 bp, PCK1, is not involved in adipocyte specificity b ecause inactivation of this site did not affect transgene expression, Moreo ver, electrophoretic mobility shift experiments indicated that, unlike PCK2 , PCK1 is not selective for PPAR gamma/RXR-binding. To characterize the enh ancer further, the rat and human PEPCK 5'-flanking DNA sequences were compa red by computer and found to have significant similarities in the enhancer region. This high level of conservation suggests that additional transcript ion factors are probably involved in enhancer function. A putative human PC K2 element was identified by this sequence comparison. The human and rat PC K2 elements bound PPAR gamma/RXR with the same affinities. This work provid es the first in vivo evidence that the binding of PPAR gamma to its target sequences is absolutely required for adipocyte-specific gene expression.