6484 Nucleic Acids Research, 2006, Vol. 34, No. 22 Consistent with these results, Luciakova et al. (54) have recently demonstrated that mutation of the NFI sites present in the promoter of the adenine nucleotide translocase-2 gene (ANT2) totally annihilated the growth-arrest properties of NFI in NIH3T3 cells. Most interestingly, they attributed these growth-arrest properties of NFI to the NFI-A, -C and -X isoforms but not NFI-B as the former were all found to repress expression of an ANT2-driven reporter gene con- struct, whereas NFI-B activated it. Members from the NFI family can either repress or activate their target genes depending on the cell type or promoter context. This means that although all NFI isoforms were found to repress to vary- ing degrees the transcription directed by the p21 promoter in HCT116 cells, yet they may activate transcription of other genes in the same cell that may or may not be related to cell growth. For instance, NFI has been shown to activate expression of p53 (55) and gadd153 (56), both of which play key roles in growth arrest of damaged or environmen- tally stressed cells. Paradoxically, NF1-X1 was identiﬁed as one of three genes (together with c-myc and MDM2) that pre- vent TGFb-induced growth arrest (57). Therefore, while all NFI isoforms clearly repress p21 gene expression in HCT116 cells, NFI-B might as well regulate expression of additional, cell-growth related genes that are not under the inﬂuence of the other NFI isoforms. This differential property of NFI-B may rely on its ability to recruit either co-repressors or co- activators that are distinct from those recruited by other NFI proteins. For instance, of all the NFI isoforms, only NFI-B has been found to activate whey acidic protein (WAP) gene expression in cooperation with STAT5A and the gluco- corticoid receptor (GR) in JEG-3 cells (58). Most interest- ingly, NFI-B deﬁcient mice have been shown to die early after birth and display severe lung hypoplasia (59), lung development being arrested at the late pseudoglandular stage. These results suggest that NFI-B is most likely required for normal lung cell proliferation in order to ensure proper lung development. It is noteworthy that the in vivo footprint identiﬁed in the present study extends 10 bp beyond the 30 end of the NFI consensus site (from 148 to 138). A search conducted using databases for the identiﬁcation of transcription factor target sites failed to identify any known nuclear protein that may interact with the 148/ 138 p21 sequence. The presence of additional co-factors that can bind DNA in vivo but not in vitro, as well as structural events, such as DNA folding, could explain the detection of the pro- tected residues located downstream from the 161 to 149 NFI site. Indeed, the RNAi experiments conducted in this study (Figure 7C) suggested that other DNA binding proteins might contribute to the appropriate transcription of the p21 gene by interacting somewhere along the 124/ 148 seg- ment from the p21 promoter as the p21–124 construct did not mimic levels achieved with the p21–192 that bears muta- tions in the 161/ 149 NFI site. Besides, reducing the endogenous level of NFI through RNAi is also likely to con- siderably alter the transcription of many genes in the trans- fected cells. Some of them may as well encode transcription factors other than NFI that also participate to the cascade of regulatory events required to ensure appropri- ate transcription of the p21 gene. Further works will be required in order to determine the precise identity of such cis- acting transcriptional regulators and whether their regulatory inﬂuences are mediated through their interaction with the 148/ 138 area from the p21 promoter. The NFI family of transcription factors is made-up of four protein subtypes (NFI-A, -B, -C and -X) (60), each encoded by a different gene, that can form either homo- or het- erodimers. The large diversity of this family of proteins, for which 19 isoforms have been described to date [reviewed in (61)], rely on the fact that each individual NFI mRNA tran- script is subjected to alternative splicing (33,62). Members from the NFI family have been shown to function either as repressors (35,46,56) or activators (63,64) of gene transcrip- tion. Through interactions with weak binding sites, NFI may also regulate gene expression by its ability to either cooperate or compete with many transcription factors (35,65). Con- sidering the negative inﬂuence exerted by NFI on p21 gene transcription, we propose that NFI may act in cooperation with other nearby co-activators or transcription factors to restrict the expression of the p21 gene throughout the cell cycle progression while DNA is undamaged. NFI was reported to interact with and antagonize Sp1, which result in the down-regulation of the platelet-derived growth factor (PDGF)-A gene expression (65). Interestingly, in vivo foot- printing analysis of the p21 promoter also revealed DNA– protein interactions at four of the six putative consensus binding sites reported for Sp1 (4). Besides NFI and Sp1, these in vivo analyses also identiﬁed protections at target sites for nuclear proteins, such as E2F1 (Figure 1) that have already been reported to bind the p21 proximal promoter. Members from the E2F family of transcription factors play a crucial role in the transactivation of G1/S transition speciﬁc genes (66). Previous studies already demonstrated the involvement of E2F1 in the p53-independent activation of p21 (43,44). Results from the ChIP analyses revealed variations in the pattern of p21 promoter occupancy by the transcription fac- tors Sp1, NFI and E2F1 between actively growing and serum-starved, quiescent HSFs. Indeed, whereas both Sp1 and NFI but not E2F1 substantially bind the p21 promoter in proliferating cells, HSFs grown under serum deprivation completely lost p21 promoter recognition by both Sp1 and NFI but gained binding of E2F1. The relative occupancy of the p21 promoter by NFI in vivo clearly exceeded that of both Sp1 and Sp3 in proliferating HSFs, thereby favoring repression rather than activation of p21 transcription. On the contrary, the lack of any NFI binding in serum-starved cells combined to the recognized positive inﬂuence of both Sp3 and E2F1 as both factors were shown to bind the p21 promoter under this condition, is consistent with the increased expression of p21 reported when cells progress toward growth arrest (44,67). Interestingly the presence of Sp3, whose binding to the p21 promoter does not change with alterations in the proliferative state of the cells, raised the interesting possibility that most of the Sp1 target sites identiﬁed in the p21 promoter might became occupied by Sp3 under serum deprivation as both transcription factors have been reported to bind the same GC-rich target sites (67). It is noteworthy that Sp1/Sp3 share extensive structural and sequence homology (68), and often cooperate in activating gene transcription (69–71). The presence of Sp3 in HSFs grown with or without serum suggests that p21 is constitutively activated by this