Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.
The GRU (glucocorticoid-response unit) within the distal enhancer of the gene encoding carbamoyl-phosphate synthase, which comprises REs (response elements) for the GR (glucocorticoid receptor) and the liver-enriched transcription factors FoxA (forkhead box A) and C/EBP (CCAAT/enhancer-binding protein), and a binding site for an unknown protein denoted P3, is one of the simplest GRUs described. In this study, we have established that the activity of this GRU depends strongly on the positioning and spacing of its REs. Mutation of the P3 site within the 25 bp FoxA-GR spacer eliminated GRU activity, but the requirement for P3 could be overcome by decreasing the length of this spacer to < or =12 bp, by optimizing the sequence of the REs in the GRU, and by replacing the P3 sequence with a C/EBPbeta sequence. With spacers of < or =12 bp, the activity of the GRU depended on the helical orientation of the FoxA and GR REs, with highest activities observed at 2 and 12 bp respectively. Elimination of the 6 bp C/EBP-FoxA spacer also increased GRU activity 2-fold. Together, these results indicate that the spatial positioning of the transcription factors that bind to the GRU determines its activity and that the P3 complex, which binds to the DNA via a 75 kDa protein, functions to facilitate interaction between the FoxA and glucocorticoid response elements when the distance between these transcription factors means that they have difficulties contacting each other.
Schoneveld
Onard J L M
OJ
AMC Liver Center, Academic Medical Center, University of Amsterdam, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.
Gaemers
Ingrid C
IC
Das
Atze T
AT
Hoogenkamp
Maarten
M
Renes
Johan
J
Ruijter
Jan M
JM
Lamers
Wouter H
WH
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D005938:Glucocorticoids / Q000235:genetics*; D051541:Hepatocyte Nuclear Factor 3-alpha; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics
D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C496501:Foxa1 protein, rat; D005938:Glucocorticoids; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D011506:Proteins; D014157:Transcription Factors; D002222:Carbamoyl-Phosphate Synthase (Ammonia)