Nucleic Acids Research, 2006, Vol. 34, No. 22 6479 Figure 3. In vitro DMS and DNase I footprinting of NFI binding to the p21 promoter. (A) The 50 end-labeled 83 bp SmaI–HindIII fragment from the p21–192 plasmid that covers p21 promoter sequences from 110 to 192 was incubated with 2 mg nuclear proteins from a CM-Sepharose-enriched preparation of rat liver NFI in the presence of either no (C; lanes 2 and 5) or a 150-fold molar excess of unlabeled competitor oligomers (NFI or Sp1; lanes 3 and 4, respectively) Formation of DNA–protein complexes was then monitored by EMSA on a 4% native polyacrylamide gel. The position of the NFI/p21–192 DNA–protein complex is indicated (NFI). The 110/ 192 labeled probe was also incubated with 2 ml of the NFI Ab to monitor the formation of the NFI/NFIAb/p21 protein– protein–DNA supershifted complex (SC in lane 6). P: labeled probe with no added protein (lane 1). U: unbound fraction of the labeled probe. (B) The labeled probe used in A was methylated with DMS and incubated with CM-Sepharose-enriched NFI before separation of the DNA–protein complex by EMSA. Both the labeled DNA from the NFI complex (NFI in panel A) and the unbound fraction of the probe (U in panel A) were isolated and further treated with piperidine before being analyzed on a 8% polyacrylamide sequencing gel. The DNA sequence from the p21 promoter that includes the protected G residues (full and half circles correspond to fully and partly protected G residues, respectively) is indicated along with its positioning relative to the p21 mRNA start site. The p21 NFI target site is also indicated (box). (C) A 228 bp DNA fragment spanning p21 promoter sequences from position 192 to +36 was 50 end-labeled and incubated with 75 mg CM-Sepharose-enriched NFI before being digested with DNase I. The position of the NFI site protected from digestion by DNase I is shown (shaded box) along with that of the p21 NFI site identified by in vivo footprinting (full line box). G: Maxam and Gilbert ‘G’ sequencing ladder; C: labeled DNA digested by DNase I in the absence of proteins. used to monitor cell cycle progression of the doxo-growth- arrested cells by FACS analyses. Transfection of HCT116 cells with the NFI expression plasmids revealed that all four NFI isoforms could reduce the activity of the p21–192 construct (up to 3.5-fold repression with NFI-X; Figure 5B). Furthermore, co-transfection of p21–192 along with the combination of NFI-B, -C and -X brought down p21 promoter function further to a 5-fold repression. Cultur- ing HCT116 cells in the presence of doxorubicin indeed arrested the cells primarily into the G2-phase of the cell cycle, with 3% of the cells in the S-phase (compare 1st and 2nd columns on Figure 5C) but did not affect the negative regulatory inﬂuence of the NFI isoforms on the activity of the p21 promoter (results not presented). Interest- ingly, while over-expressing NFI-A, -C and -X substantially reduced the proportion of the cells committed into growth arrest (down to 1% with NFI-A), over-expression of NFI-B considerably increased those in the S-phase (up to 9%). Transfection of doxorubicin-treated HCT116 cells with the combination of the NFI-B, -C and -X dramatically reduced the cells remaining in S-phase from 2.7 ± 2.0 to 0.4 ± 0.7.