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Nucleic Acids Research 2006 34(2):543-554; doi:10.1093/nar/gkj460
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Published online 24 January 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Regulation of the human p21(waf1/cip1) gene promoter via multiple binding sites for p53 and the vitamin D3 receptor

Anna Saramäki1, Claire M. Banwell1,2, Moray J. Campbell2 and Carsten Carlberg1,*

1Department of Biochemistry, University of Kuopio FIN-70211 Kuopio, Finland 2Division of Medical Sciences, Institute of Biomedical Research, University of Birmingham Medical School Edgbaston, Birmingham, B15 2TH, UK

*To whom correspondence should be addressed. Tel: +358 17 163062; Fax: +358 17 2811510; Email: carlberg{at}messi.uku.fi

Received October 7, 2005. Revised December 11, 2005. Accepted January 5, 2006.

The main regulator of the human tumor suppresser gene p21(waf1/cip1) is the transcription factor p53, but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor VDR in the presence of its ligand 1{alpha},25-dihydroxyvitamin D3 (1{alpha},25(OH)2D3). To identify VDR responding regions, we analyzed 20 overlapping regions covering the first 7.1 kb of the p21(waf1/cip1) promoter in MCF-7 human breast cancer cells using chromatin immuno-precipitation assays (ChIP) with antibodies against p53 and VDR. We confirmed two known p53 binding regions at approximate positions –1400 and –2300 and identified a novel site at position –4500. In addition, we found three VDR-associated promoter regions at positions –2300, –4500 and –6900, i.e. two regions showed binding for both p53 and VDR. In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53-positive promoter regions and also five 1{alpha},25(OH)2D3 response elements within the three VDR-positive regions. Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5-fluorouracil and 1{alpha},25(OH)2D3. Moreover, re-ChIP assays confirmed the functionality of the three 1{alpha},25(OH)2D3-reponsive promoter regions by monitoring simultaneous occupancy of VDR with the co-activator proteins CBP, SRC-1 and TRAP220. Taken together, we demonstrated that the human p21(waf1/cip1) gene is a primary 1{alpha},25(OH)2D3-responding gene with at least three VDR binding promoter regions, in two of which also p53 co-localizes.


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