6472–6487 Nucleic Acids Research, 2006, Vol. 34, No. 22 doi:10.1093/nar/gkl861 Published online 27 November 2006 Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells Ste´ phane Ouellet, Franc¸ois Vigneault1, Maryse Lessard1, Steeve Leclerc1, Re´ gen Drouin and Sylvain L. Gue´ rin1,2,* Service of Genetics, Department of Pediatrics, Universite´ de Sherbrooke, Sherbrooke, Que´ bec, Canada, 1Oncology and Molecular Endocrinology Research Center and 2Unit of ophthalmology, CHUL, Centre Hospitalier Universitaire de Que´ bec and Laval University, Que´ bec, Que´ bec, Canada Received September 6, 2006; Revised September 29, 2006; Accepted October 3, 2006 ABSTRACT The cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21 (WAF1/CIP1) modulates cell cycle, apoptosis, senescence and differentiation via spe- cific protein–protein interactions with the cyclins, cyclin-dependent kinase (Cdk), and many others. Expression of the p21 gene is mainly regulated at the transcriptional level. By conducting both ligation-mediated PCR (LMPCR) and chromatin immunoprecipitation (ChIP) in vivo, we identified a functional target site for the transcription factor, nuclear factor I (NFI), in the basal promoter from the p21 gene. Transfection of recombinant constructs bearing mutations in the p21 NFI site demonstrated that NFI acts as a repressor of p21 gene expression in various types of cultured cells. Inhibition of NFI in human skin fibroblasts through RNAi considera- bly increased p21 promoter activity suggesting that NFI is a key repressor of p21 transcription. Over- expression of each of the four NFI isoforms in HCT116 cells established that each of them con- tribute to various extend to the repression of the p21 gene. Most of all, over-expression of NFI-B in doxorubicin, growth-arrested HCT116 increased the proportion of cells in the S-phase of the cell cycle whereas NFI-A and NFI-X reduced it, thereby estab- lishing a role for NFI in the cell cycle dependent expression of p21. INTRODUCTION p21 (CDKN1A) belongs to a family of cell-cycle dependent kinase inhibitors. p21 modulates various processes such as cell growth (1), differentiation (2) and apoptosis (3). DNA-damaging agents such as UV-irradiations or carcino- gens induce the transcriptional activation of p21 via a p53- dependent pathway. Phosphorylated p53 binds to regulatory elements present in distal regions of the p21 promoter and transactivates p21 transcription via physical and functional interactions with the ubiquitous transcription factor Sp1 bound to the proximal region (4,5). Strong p21 accumulation lead to suppression of cdk activity, allowing the accumulation of hypophosphorylated Rb, inhibition of E2F-dependent tran- scriptional processes, and cell cycle arrest in G1 (6–8). By interacting directly with PCNA, an auxiliary factor for DNA polymerases d and e, p21 also prevents DNA synthesis and regulates DNA methylation without alteration of the PCNA-dependent nucleotide-excision repair (9,10). Some reports also suggest a p53-independent activation of p21 following UV-irradiation (11,12) but the mechanisms of induction remained poorly understood. Inducers of differen- tiation such as steroid hormones (13), transforming growth factor-b (TGF-b), phorbol ester or phosphatase inhibitors (14), interferon g (15), progesterone (16), nerve growth factor (17) and platelet-derived growth factor (18) enhance p21 gene expression in many different cell systems. As for p53, the majority of these modulators affect p21 gene transcription via Sp1 and Sp3 interactions at the proximal promoter. Novel alternate p21 transcripts that are upregulated as a result of DNA damage-induced p53 activation and are dependent on p53 for their basal or induced expression were recently discovered (19,20). In proliferating cells, p21 is expressed at a basal level in a constitutive and cell cycle dependant way (21). Under these conditions, most of the p21 proteins are components of the cyclin/cdk active complex (22). Therefore, it is likely that p21, when expressed at a moderate level, can act as an anchor protein as well as an assembly factor for cyclin D-cdk4/cdk6 thereby promoting their mutual interactions and consequently cell cycle progression, which is in complete contrast to its function as a cdk inhibitor (23). However, there seems to *To whom Correspondence should be addressed. Tel: +418 654 2296; Fax: +418 654 2761; Email: sylvain.guerin@crchul.ulaval.ca The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.