C/EBPbeta interacts with the P-enolpyruvate carboxykinase adipocyte-specific enhancer.
CCAAT/enhancer binding protein (C/EBP) family members are known to transactivate 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 homozygous null for C/EBPbeta cannot express PEPCK when induced to differentiate into adipocytes (Tanaka et al., EMBO J. 16, 7432-7443, 1997). This along with our previous observation that an upstream adipocyte-specific enhancer contains multiple putative C/EBP binding elements suggested the possibility that C/EBPbeta transactivates the PEPCK gene in adipocytes via distal elements. We report here that C/EBPbeta transactivates a PEPCK-luciferase chimera in transient transfection assays. C/EBPbeta acted independently of peroxisome proliferator-activated receptor gamma (PPARgamma) which is required for function of the enhancer. C/EBPbeta in nuclear extracts and recombinant C/EBPbeta bound three of the putative C/EBP-binding elements within the enhancer. C/EBPbeta binding to these three elements was strongly cooperative. However, mutation of all three elements did not affect reporter transactivation by C/EBPbeta suggesting that additional elements participate in PEPCK regulation or that the effects of C/EBPbeta are indirect.
Eubank
D W
DW
Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
Williams
S C
SC
Beale
E G
EG
D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000187:drug effects / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D014162:Transfection
D022782:CCAAT-Enhancer-Binding Protein-beta; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D010729:Phosphoenolpyruvate Carboxykinase (GTP)