In vivo footprinting of the carbamoylphosphate synthetase I cAMP-response unit indicates important roles for FoxA and PKA in formation of the enhanceosome.
The expression of carbamoylphosphate synthetase-I (CPS), the first and rate-determining enzyme of the urea cycle, is regulated at the transcriptional level by glucocorticoids and glucagon, the latter acting via cyclic AMP (cAMP). The hormonal response is mediated by a distal enhancer located 6.3 kb upstream of the transcription-start site. Within this enhancer, a cAMP-response unit (CRU) is responsible for mediating cAMP-dependent transcriptional activity. The CPS CRU contains binding sites for cAMP-response element (CRE)-binding protein (CRE-BP), forkhead box A (FoxA), CCAAT/enhancer-binding protein (C/EBP), and an unidentified protein P1. To gain insight in the protein-DNA interactions that activate the CPS CRU in living cells, we have employed in vivo footprinting assays. Comparison of the fibroblast cell line Rat-1 and the hepatoma cell lines FTO-2B and WT-8 showed that FoxA binds the CPS CRU constitutively in CPS-expressing cells only. Comparison of FTO-2B and WT-8 hepatoma cells, which only differ in cAMP responsiveness, demonstrated that the binding of the other transcription factors is dependent on cAMP-dependent protein kinase (PKA) activity. Finally, we observed a footprint between the CRE and the P1-binding site in the in vivo footprint assay that was not detectable by in vitro footprint assays, implying a major change in CRU-associated chromatin conformation upon CRU activation. These findings indicate that activation of the CRU is initiated in a tissue-specific manner by the binding of FoxA. When cellular cAMP and glucocorticoid levels increase, CRE-BP becomes activated, allowing the binding of the remaining transcription factors and the transactivation of the CPS promoter.
Hoogenkamp
M
M
AMC Liver Centre, Academic Medical Centre, University of Amsterdam, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.
Stallen
J M P
JM
Lamers
W H
WH
Gaemers
I C
IC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018983:DNA Footprinting; D004742:Enhancer Elements, Genetic; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D051381:Rats; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D051858:Forkhead Transcription Factors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002222:Carbamoyl-Phosphate Synthase (Ammonia)