Nucleic Acids Research, 2006, Vol. 34, No. 22 6481 Figure 5. Influence of NFI over-expression on p21 promoter activity in growth-arrested HCT116 cells. (A) Crude nuclear extracts were prepared from both HCT116 and HeLa cells (used as a control) and examined for expression of Sp1 and NFI in Western blot analyses. (B and C) The p21–192 recombinant construct was co-transfected with the empty vector pCH (+EV), or with expression plasmids encoding each of the NFI isoforms, either individually (+NFI-A, -B, -C and -X) or in combination (+NFI-BCX), in doxorubicin, growth-arrested HCT116 cells. Cells were collected 48 h later and used either for the measurement of the CAT activity (panel B) or to determine the proportion of the cells engaged in the S-phase of the cell cycle by FACS analyses (panel C). endogenous NFI transcript by RNAi (Figure 7C). Because HeLa cells are much easier to culture and are transfected with a higher efﬁciency, they were selected over HSFs for con- ducting the RNAi assays. Recombinant constructs bearing vari- ous lengths from the p21 promoter (up to 50 positions 124, 192 and 2300) were co-transfected with and without a com- bined pool of siRNAs directed against NFI-A, -B, -C and -X. As expected, the P21–124 construct lacking the NFI binding site yielded a promoter activity higher than the NFI-bearing con- structs p21–192 and p21–2300, which are both under the nega- tive regulatory inﬂuence of NFI. Co-transfection of these constructs along with the NFI siRNAs substantially released repression by NFI for both p21–192 (3-fold increase) and p21–2300 (near 7-fold increase). Again, mutating the NFI bind- ing site in p21–192 (p21–192 mNFI) strongly increased CAT activity in HeLa cells (by 8-fold). However, while inhibition of endogenous NFI through RNAi considerably increased the activity of the unmutated, wild-type p21–192 construct (3-fold increase) it had no such inﬂuence when the NFI site is mutated in p21–192 mNFI (which yielded a weak 27% reduction in p21 promoter activity). DISCUSSION Cyclin-dependent kinase inhibitor p21 plays a key function in cell cycle arrest at the G1/S checkpoint in response to DNA damage, and is involved in the assembly of active cyclin- kinase complexes especially cyclin D-Cdk4/6. Transcription of the p21 gene relies on the control of numerous regulators of which many still have to be identiﬁed. In the present study, we precisely mapped, through both in vivo (LMPCR) and in vitro (DMS and DNase I footprinting) approaches, a target site for the transcription factor NFI in the basal promoter of the human p21 gene that proved to function as a powerful repressor of p21 gene transcription in both non-damaged and proliferating primary cultures of human ﬁbroblasts and established tissue culture cells. To our knowledge, no evidence regarding a regulatory function exerted by NFI on the transcription of the p21 gene has ever been published until now. We demonstrated that members of the NFI family could bind a target site on the p21 basal promoter located between positions 161 and 149, and considerably repress its transcription in a variety