6478 Nucleic Acids Research, 2006, Vol. 34, No. 22 consensus sequence located from positions 161 to 149 in primary cultured HSFs. The NFI target site from the p21 promoter differs from the prototypical sequence by 3 nucleotides over the entire 15 bp that constitutes this site (Figure 2A). Recently, Roulet et al. (48) conducted a very elegant study in which they function- ally validated a binding site predictor that could predict the afﬁnity of a degenerated NFI site relative to that of the prototypical NFI sequence. According to their mathematical model, NFI is expected to bind the p21 NFI target site with an afﬁnity of over 91% that normally yielded when it binds the high-afﬁnity NFI prototypical (consensus) sequence (which, in this case, is considered to correspond to an afﬁnity of 100%). We therefore assessed the afﬁnity of NFI toward both the p21 and the prototypical NFI target sites by incu- bating crude nuclear proteins from HSFs with the p21.1 labeled probe in the presence of varying concentrations (5- to 500-fold molar excesses) of either unlabeled p21.1 or the NFI consensus oligomer. As shown on Figure 2C, the unlabeled p21.1 oligomer was as efﬁcient as the prototypical NFI-bearing oligonucleotide in competing for the formation of the NFI complex in EMSA. This result provided evidence that NFI binds to the p21 degenerated NFI site with an afﬁn- ity undistinguishable from that obtained with the prototypical NFI site. Recognition of DNA target sites by transcription factors might on occasion differ depending on whether the labeled probe used in the EMSA is synthetic (as the double-stranded oligonucleotide p21.1 used above) or derived from the gene’s regulatory sequence. To further validate the results from the EMSAs conducted using the p21.1 oligonucleotide, EMSA experiments were repeated by using an 83 bp genomic frag- ment obtained from the SmaI–HindIII digestion of the p21– 192 plasmid and covering the p21 promoter sequence from positions 110 to 192 as labeled probe. For this speciﬁc purpose, and to increase the sensitivity of the assay, we used a carboxymethyl (CM)-Sepharose enriched preparation of rat liver NFI-L (29) as the source of NFI protein. As shown on Figure 3A (lane 2), NFI-L indeed bound to the 110/ 192 p21 labeled probe very efﬁciently. Speciﬁcity for the formation of the NFI complex was further demon- strated by both competition experiments in EMSA (only the NFI but not the unrelated Sp1 competitor oligonucleotide could compete for the formation of the shifted complex; Figure 3A, lanes 3 and 4, respectively) and supershift experi- ments [Figure 3A; addition of the NFI Ab (lane 6) super- shifted the NFI complex from its normal position on the gel (NFI) to that corresponding to a complex with low elec- trophoretic mobility in gel (SC)]. We next performed both DMS methylation interference assays and DNase I footprinting in vitro in order to more pre- cisely deﬁne the DNA target site bound by NFI along the 110 to 192 p21 promoter fragment. As shown in Figure 3B, methylated G residues that interfere with binding of NFI to its target site in the p21 promoter in vitro also per- fectly mapped within the site protected by NFI and deﬁned through the use of the in vivo footprinting procedures (see Figure 1). In vitro DNase I footprinting identiﬁed only a short stretch of protected sequence ( 3 bp) that is contained within the NFI site identiﬁed in vitro by DMS footprinting and in vivo by LMPCR. The lack of DNase I break points in the binding site of NFI, as revealed by the absence of bands on the autoradiogram, did not allow the determination of the entire sequence protected by the protein. This in vitro result contrasts with the in vivo footprinting data for this particular p21 promoter region as a larger protection could be observed (extending over 14 bp; see Figure 1). We there- fore conclude that NFI indeed possesses the ability to bind efﬁciently to the p21 basal promoter both in vivo and in vitro by interacting with a target site located between positions 161 and 149. NFI functions as a repressor of p21 promoter activity and triggers G2-arrested cells into the S-phase of the cell cycle In order to assess the regulatory inﬂuence exerted by the p21 NFI site, DNA fragments spanning the p21 basal promoter and extending from 50 position 192 up to either 30 positions +36 (which include the NFI site) or –124 to +36 (in which the NFI site has been deleted) was inserted upstream the CAT reporter gene (Figure 4A) and transiently transfected into either primary cultured HSFs or tissue culture cells (HeLa and GH4C1). Deletion of the entire NFI site identiﬁed by in vivo footprinting (in p21–124), yielded a signiﬁcant increase in the activity of the CAT reporter gene upon trans- fection of the three different types of cells used in this study (3.1-, 2.9- and 2.3-fold increase in CAT activity relative to the level directed by the p21–192 plasmid in HSFs, HeLa and GH4C1 cells, respectively; Figure 4B). In order to prevent undesirable regulatory effects that might have resulted from the deletion of sequences nearby the p21 NFI site in the p21–124 construct, the NFI site from the parental plasmid p21–192 was also altered through site-directed mutagenesis. The ﬁve residues identiﬁed through both DMS methylation interference and LMPCR footprinting as the most important for NFI binding out of the 15 bp that constitute the p21 NFI site were changed for As and Ts (the wild-type NFI site 50-CTGGAACTCGGCCAG-30 was changed to 50-CTaa- AACTCGattAG-30) to create the NFI-mutated construct p21–192 mNFI. Consistent with the data from the deletion analysis, mutation of the p21 NFI site in p21–192 mNFI also yielded a substantial increase in CAT activity relative to the unmutated, wild-type NFI-bearing construct p21–192 upon transfection of all cell types (8.5-, 2.3- and 3.1-fold increase in HSFs, HeLa and GH4C1 cells, respectively), further validating the results from the deletional analysis (Figure 4B). To further demonstrate the negative regulatory inﬂuence of NFI on the p21 promoter, p21–192 was co-transfected along with expression plasmids encoding high level of expression of each of the NFI isoforms into HCT116 cells. This cell line proved to be particularly interesting as it expresses very little NFI protein in Western blot analyses, unlike Sp1, which is abundantly expressed in these cells (Figure 5A). Because HCT116 cells can be easily growth arrested in the G2-phase of the cell cycle by the addition of doxorubucin, p21–192 was therefore transfected either alone or in the presence of each of the NFI expression plas- mid (NFI-A, -B, -C and -X) in growth-arrested HCT116 cells. Part of the transfected HCT116 cells were used for the mea- surement of the CAT activity while the remaining cells were