be a lack of understanding on the regulatory mechanisms involved in this basal expression of the p21 gene. In this study, we provided evidence that NFI is a major contributor of p21 gene expression as it could repress its transcription by interacting with the p21 proximal promoter in vivo. MATERIALS AND METHODS Cell culture Human skin ﬁbroblasts (HSFs) were isolated from skin biops- ies of healthy male donors and primary cultured in DMEM (Gibco BRL, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (FBS, Gibco BRL). Human epith- eloid carcinoma HeLa cells (ATCC CCL 2) were grown in DMEM supplemented with 10% FBS. Rat pituitary GH4C1 cells were grown in Ham’s F10 medium (Sigma-Aldrich, Oakville, ON, Canada) supplemented with 10% FBS. The human colorectal cancer cell line HCT116, kindly donated by Dr Chantal Guillemette (Oncology and Molecular Endo- crinology Research Center, CHUL, Que´bec, Canada), was grown in McCoy’s medium (Invitrogen cat# 16600) supple- mented with 10% FBS. All cells were grown under 5% CO2 at 37 C and gentamycin was added to all media at a ﬁnal concentration of 15 mg/ml. Cell cycle analysis HCT116 cells (2.5 · 105 cells/well) were plated into 6-wells tissue culture plates (Sarstedt, Montre´al, QC, Canada) in complete McCoy’s medium and then growth arrested by the addition of 500 nM doxorubicin (Sigma-Aldrich). Approxi- mately 48 h later, cells were collected, ﬁxed with 70% cold ethanol, and stored at –20 C overnight. Fixed cells were washed with PBS and incubated with PBS containing RNase (100 mg/ml) and PI (10 mg/ml) at room temperature in the dark for 30 min. The DNA content of the cells were analyzed using ﬂow cytometry and acquisition of data for 105 events were performed using an Epics XL ﬂow cytometer (Beckman Coulter, Miami, FL). The distribution of HCT116 cells within each phase of the cell cycle was analyzed from dual parameters histograms using an Epics Elite ﬂow cytometry workstation version 4.5 software. In vivo footprinting HSFs were seeded into 150 mm culture plates at a density of 50% cells/plate and grown as above for 3 days. Living cells and puriﬁed DNA (referred as in vivo and in vitro, respec- tively) were treated with one of the following probing agents: 0.02% dimethylsulphate (DMS; Sigma-Aldrich Canada), 2 ultraviolet C (UVC; 1500 J/m ) irradiation (G15T8 germi- cidal lamp; Philips, Montreal, QC, Canada) and DNase I (8.75 g/ml; Worthington Biochemical, Lakewood, NJ, USA) as previously described (24,25). Strand break frequencies were estimated on an alkaline agarose gel. LMPCR was carried out as previously described (24,25) using two primer sets (1A: 50-TCTCTCACCTCCTCTGA-30, position +97 to +81; 1B: 50-CTGAGTGCCTCGGTGCCTCGGCG-30, posi- tion +85 to +62; and 2A: 50-CCAGA TTTGTGGCTCAC- 30, position 280 to 264; 2B: 50CTCACTTCGGGGGAAA- TGTGTCCAGCG-30; position 268 to 241), allowing the analysis of the proximal promoter of p21 from nt 220 to Nucleic Acids Research, 2006, Vol. 34, No. 22 6473 +40. Brieﬂy, gene-speciﬁc primers were annealed to the genomic fragments of varying sizes and then extended using cloned Pfu exo-DNA polymerase (Stratagene, LaJolla, CA) to produce double-strand blunt ends. An asymmetric double-strand linker (L25: 50-GCGGTGACCCGGGAGAT- CTG-AATTC-30 and oligo L11: 50-GAATTCAGATC-30) was then ligated to the phosphorylated terminal end of each fragment, providing a common sequence on the 50 end of all fragments. Using Pfu exo-DNA polymerase, a linker- speciﬁc primer was used for a single round of linear ampli- ﬁcation, followed by PCR ampliﬁcation using the appropriate primer sets in combination with the linker primer. All primer extensions and PCR ampliﬁcations were carried out on T gra- dient thermocycler from Biometra (Montreal Biotech, Inc. Kirkland, Canada) as described (24,25). The PCR-ampliﬁed fragments were phenol/chloroform extracted, ethanol precipi- tated and subjected to electrophoresis on 8% polyacrylamide, 7 M urea gels alongside a Maxam and Gilbert sequencing ladder, followed by electrotransfer to nylon membranes (Roche Diagnostics Corp., Laval, Canada), hybridization to a 32P-labeled gene-speciﬁc probe and visualization by auto- radiography on Kodak ﬁlms (Amersham Biosciences, Baie d’Urfe´, Canada). Plasmid constructs The p21 promoter fragment spanning region 192 to +36 (p21–192) relative to the mRNA start site was produced by KpnI/BglII digestion of the plasmid p21 (0–2300)-Luc containing the entire p21 promoter (kindly provided by Dr Claude Labrie, Oncology and Molecular Endocrinology Research Center, CHUL Research Center, Que´bec, Canada). Synthetic oligomers were used to produce two distinct link- ers allowing the ligation of the fragment upstream of the chloramphenicol acetyltransferase (CAT) reporter gene in the HindIII/XbaI-linearized vector pCATbasic (Promega, Madison, WI). The plasmid lacking the region 192 to 125 (p21–124) was obtained through double-digestion of the p21–192 construct with the KpnI and BstX1 restriction enzymes. The p21–192 mNFI construct that bear a mutated NFI binding site was produced by the PCR, using p21–192 as a template and the synthetic oligomers p21-NFImA/B (50-GGACCGGCTGGCCTGCTAAAACTCGATTAGGCTC- AGCTGGCTCC-30)/(50-GGAGCCAGCTGAGCCTAATC- GAGTTTTAGCAGGCCACCGGTCC-30). PCR ampliﬁcations were performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s speciﬁcations. The DNA insert from each recombinant plas- mid was sequenced by chain-termination dideoxy sequencing (26) to conﬁrm the mutations. The pCH-NFI-A1.1, pCH-NFI-B, pCH-NFI-C and pCH-NFI-X expression plas- mids that encode high levels of the NFI-A, -B, -C and -X NFI isoforms, as well as the empty vector pCH that was used to derive the NFI expressing plasmids have been kindly provided by Dr Richard M. Gronostajski (Department of Biochemistry, SUNY at Buffalo, Buffalo, NY, USA). Protein extracts and electrophoretic mobility shift assays Nuclear extracts from primary cultured HSFs deprived or not of serum for 48–72 h, or from rat pituitary GH4C1 and HeLa