C/EBP beta interacts with the P-enolpyruvate carboxykinase adipocyte-specific enhancer

CCAAT/enhancer binding protein (C/EBP) family members are known to transact ivate the gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) in hepatocytes via promoter proximal C/EBP response elements. PEPCK is also expressed in adipocytes; however, fibroblasts that are homoz ygous null for C/EBP beta cannot express PEPCK when induced to differentiat e into adipocytes (Tanaka et al, EMBO J. 16, 7432-7443, 1997). This along w ith our previous observation that an upstream adipocyte-specific enhancer c ontains multiple putative C/EBP binding elements suggested the possibility that C/EBP beta transactivates the PEPCK gene in adipocytes via distal elem ents. We report here that C/EBP beta transactivates a PEPCK-luciferase chim era in transient transfection assays. C/EBP beta acted independently of per oxisome proliferator-activated receptor gamma (PPAR gamma) which is require d for function of the enhancer. C/EBP beta in nuclear extracts and recombin ant C/EBP beta bound three of the putative C/EBP-binding elements within th e enhancer. C/EBP beta binding to these three elements was strongly coopera tive. However, mutation of all three elements did not affect reporter trans activation by C/EBP beta suggesting that additional elements participate in PEPCK regulation or that the effects of C/EBP beta are indirect. (C) 2001 Academic Press.