Cells were then cross-linked with 1% formaldehyde for 10 min, harvested, and chromatin immunoprecipitated with 1 mg polyclonal antibodies directed against the Sp1 (sc-59; Santa Cruz Biotechnology), Sp3 (sc-644; Santa Cruz Biotech- nology), NFI and E2F1 (sc-193·; Santa Cruz Biotechnology) transcription factors (Santa Cruz Biotechnology, Inc. Santa Cruz, CA). The resulting DNA was analyzed by PCR using a pair of primers (p21–279U: 50CAGATTTGTGGCTC-AC- TTCGTGG-30 and p21+223L: 50AGAAACA-CCTGTGAA- CGCAGCAC-30) spanning the entire p21 gene promoter area from nt –279 to +223. As a negative control, each ChIP sample was also subjected to PCR using primers (p21–2587U: 50AATTCCTCTGAAAGCTGACTGCC30 and p21–2132L: 50AGGTTTACCTGGGGTCTTTA-GA-30) spe- ciﬁc to a region located 2 kbp upstream from the p21 promoter. RNAi assays SilencerTM negative control (id #4611, #4613, #4615), and SilencerTM pre-designed siRNA duplexes against NFI-A (id #115686, #115687, #144076), NFI-B (id #115688, #115689, #115690), NFI-C (id #215174, #215173) and NFI-X (id #115298, #115297, #3296) were purchased from Ambion, Inc. (Austin, TX, USA) and used according to manufacturer’s speciﬁcations. Brieﬂy, 2.5 mg of all three siRNAs directed against NFI-A mRNA were combined and transfected in triplicate into HSFs cultured to sub-conﬂuence onto 39-mm tissue culture dishes (1 · 106 cells per dish at start) by the calcium phosphate precipitation method along with 10 mg of the appropriate plasmid construct (p21–124, p21–192, p21–192-mNFI, p21–2300 and p21–2300-mNFI). Cells were harvested 48 h following the addition of fresh medium and processed as mentioned above for the CAT assay. RESULTS In vivo footprinting of the p21 gene promoter by LMPCR In an attempt to identify the transcription factors that regu- late p21 gene transcription, we performed in vivo footprinting analyses of the proximal promoter region from the p21 gene in normal, proliferating primary cultures of HSFs by exploit- ing the LMPCR procedure (24). Footprint analyses were per- formed on the p21 promoter region covering both strands from position 220 to +40 relative to the p21 mRNA start site. Many protected and hyper-reactive sites were detected along the area from the p21 promoter that bear a putative NFI target site, on both the top and bottom strands. As shown on Figure 1A to 1C (and also summarized in Figure 1D), the comparison of the band intensity from the in vivo and in vitro treatments revealed many protected and hypersensitive nucleotides. Consistent with studies that reported binding of TBP to the TATA-box of other active gene promoters in different human cells (40,41), we also observed protected residues along the TATA-box of the p21 gene (from nt 28 to 14) as shown on Figure 1B (and summarized on Figure 1D). The DNase I protection extended many base pair beyond the 30 end of the TATA- box that is typical of the association of other members of Nucleic Acids Research, 2006, Vol. 34, No. 22 6475 the holoenzyme with TBP. In addition, four of the six puta- tive Sp1 binding sites reported to be required for p21 gene expression under basal and inducible conditions [(4,42); see discussion for details] were indeed protected from methyla- tion by DMS. Two additional p21 promoter regions that revealed DNA–protein interactions were also identiﬁed by LMPCR. The ﬁrst corresponds to the DNA target site for the transcription factor E2F1 ( 84 to 80) that has already been shown to up-regulate transcription of the p21 gene [(43,44); see discussion for details]. The second protected area extends from position 161 to 138 and encompasses a potential binding site for NFI ( 161 to 149). This large DNA–protein interaction interface also extended 10 bp beyond the 30 end of the NFI site, from 148 to 138. How- ever, search using computer databases for the identiﬁcation of transcription factors target sites could not suggest any known putative transcription factor that could interact with this speciﬁc DNA segment from the p21 promoter. Proteins from the NFI family bind the 161/ 149 sequence of the p21 promoter Both competition and supershift experiments in EMSA were conducted to demonstrate whether NFI possesses the ability to bind the 161/ 149 region from the p21 gene (Figure 2). Incubation of a labeled probe (p21.1) bearing the entire footprinted region from positions 167 to 127 (Figure 2A), with nuclear proteins from primary cultured HSFs (Figure 2B) yielded a diffuse pattern of DNA–protein complexes (lane 2) identical to that reported previously for NFI (45–47). The formation of these DNA–protein com- plexes was almost completely abolished by the addition of a 150-fold molar excess of an unlabeled competitor oligo- nucleotide bearing the consensus sequence for CTF/NFI (lane 8). Formation of these complexes was also prevented when either the p21.1 (lane 3) or the p21.2 double-stranded oligonucleotides (lane 4), both of which has the NFI-binding site from the p21 promoter (Figure 2A), were used as com- petitors (Figure 2B). However, the p21.3 oligonucleotide, which is deleted from the NFI binding site (Figure 2A), was unable to compete for the formation of the p21.1/protein complexes (data not shown). This result suggests that the 127 to 144 p21 sequence, which bear the protected resi- dues identiﬁed immediately 30 from the NFI site in the p21 promoter, is not required for NFI binding and is likely recognized by a yet unknown nuclear protein. Derivatives of the p21.1 oligonucleotide that bear mutations either in the 50 (in p21.1m50) or the 30 (in p21.1m30) half-site of NFI could still compete, although not as efﬁciently as the wild-type p21.1 oligomer, for NFI binding to the labeled probe (28% reduction with p21.1m50 (lane 5) and 43% with p21.1m30 (lane 6) as evaluated using Biorad Quantity One software) indicating that NFI possesses the ability to bind to each single half-site from the p21 NFI site in vitro. The oligomer bearing both mutated sites (p21.1m5030) was unable to compete for the formation of this complex in EMSA (lane 7). Competitors bearing the target sequences for the unrelated transcription factors AP1 (lane 9) and Sp1 (lane 10) were unable to compete for the formation of the shifted complexes yielded by the HSFs nuclear extract, a further evidence that the complexes detected