6476 Nucleic Acids Research, 2006, Vol. 34, No. 22 Figure 1. Genomic footprinting of the human p21 gene promoter. The region shown was analyzed with primer set 1, to reveal the bottom strand sequence from nt 167 to 85 (A), and the primer set 2, to reveal the upper strand sequence from nt 114 to 12 (B) and nt 167 to 137 (C) relative to the transcription initiation site. Lane 1, 8 and 10: LMPCR of naked DNA purified from primary cultures of HSFs treated in vitro (t) with DMS (lane 1), UVC (lane 8) or DNase I (lane 10). Lanes 2, 7 and 9: LMPCR of DNA purified from HSFs treated in vivo (v) with DMS (lane 2), UVC (lane 7) or DNase I (lane 9) prior to DNA purification. Lanes 3–6: Maxam-Gilbert sequencing. DMS protected and hypersensitive guanines are indicated by opened and closed circles, respectively, on each side of the autoradiograms, whereas UVC protected and hypersensitive sites are indicated by opened and closed squares. The DNase I protected and hypersensitive sites are indicated by and +, respectively. (D) Summary of the in vivo DMS, UVC and DnaseI footprints identified along the 187 to 136 human p21 gene promoter. The position of the consensus sequence for the specified transcription factors is also indicated above the sequence. indeed result from the recognition of the p21.1 labeled probe by NFI. To validate the identity of NFI as the transcription factor binding the p21.1 labeled probe, supershift experiments were conducted using a polyclonal Ab directed against an N-terminal epitope shared by all members of the NFI family. The NFI Ab recognized at least one of the proteins binding the p21.1 probe and led to the formation of a larger supershifted complex (SC in Figure 2B, lane 12). This new complex did not result from the non-speciﬁc recognition of the labeled probe by the NFI Ab or by an unrelated serum protein as neither condition (Figure 2B, lanes 13 and 14) could yield the supershifted complex seen when the NFI Ab is incubated with the p21.1 probe in the presence of nuclear proteins (Figure 2B, lane 12). Altogether, these results suggest that NFI binds the p21 promoter through its