Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.

O.J. Schoneveld, I.C. Gaemers, A.T. Das, M. Hoogenkamp, J.W. Renes, J. Ruijter, W. Lamers

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Abstract

Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.

Schoneveld OJ, Gaemers IC, Das AT, Hoogenkamp M, Renes J, Ruijter JM, Lamers WH.

AMC Liver Center, Academic Medical Center, University of Amsterdam, Meibergdreef 69-71, 1105 BK, Amsterdam, The Netherlands.

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.
Original languageEnglish
Pages (from-to)463-470
Number of pages7
JournalBiochemical Journal
Volume382
Issue numberPt 2
DOIs
Publication statusPublished - 1 Jan 2004

Cite this

Schoneveld, O. J., Gaemers, I. C., Das, A. T., Hoogenkamp, M., Renes, J. W., Ruijter, J., & Lamers, W. (2004). Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene. Biochemical Journal, 382(Pt 2), 463-470. https://doi.org/10.1042/BJ20040471