Figure 3.

In vitro DMS and DNase I footprinting of NFI binding to the p21 promoter. (A) The 5′ 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 μg 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 μl 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 5′ end-labeled and incubated with 75 μg 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.