6480 Nucleic Acids Research, 2006, Vol. 34, No. 22 Figure 4. Transient transfection analysis. (A) Schematic representation of the plasmids used. The NFI target site from the p21 promoter is shown. Numbers indicate positions relative to the p21 mRNA start site. (B) The plasmids shown in A were transfected into HSFs, as well as in the tissue culture cell lines GH4C1 and HeLa. Cells were harvested and CAT activities determined and normalized to secreted hGH. *CAT activities that are statistically different from those obtained with the p21–192 construct (P < 0.05; paired samples, t-test). Serum starvation alters the NFI-mediated repression of p21 gene transcription P21 being cell cycle regulated, we investigated the NFI inﬂuence on p21 gene transcription upon transfection of both the wild-type p21–192 construct or its NFI-mutated derivative p21–192 mNFI into primary HSFs grown in serum-free medium, a condition under which p21 is known to be highly expressed (49–51). As expected, basal p21 pro- moter activity increased considerably upon serum starvation (about 3-fold for p21–192 and 4-fold for p21–192 mNFI; Figure 6A). Under the experimental conditions used to con- duct this transfection, which differ slightly from those used above in Figure 4 (see Experimental Procedures), mutation of the NFI site yielded an 11-fold increase in CAT activity when HSFs were maintained in complete medium. However, p21 promoter activity increased further up to 17-fold in serum-starved HSFs. This increase in p21 promoter activity when cells are maintained under serum-free condition corre- lated with a signiﬁcant decrease in NFI binding in EMSA (Figure 6B) when ﬁbroblasts are deprived of serum for vari- ous periods of time (24, 48 and 72 h), despite that no altera- tion in the absolute amount of the NFI protein is observed in Western blot (Figure 6C). Indeed, while extracts from HSFs grown in the presence of serum yielded a very clear and intense NFI-labeled probe DNA–protein complex in the EMSA (Figure 6B, lane 2), culturing them under serum star- vation for various periods of time (48, 60, 66 and 72 h) resulted in a more diffused, strongly reduced NFI signal (Figure 6B, compare lanes 4, 6, 8 and 10 with lane 2). This reduction in NFI binding could not be accounted for from the degradation of NFI as no signiﬁcant quantitative nor qualitative changes are observed in the total amount of NFI as revealed by Western blot (Figure 6C). Besides, maintain- ing HSFs under serum-free condition up till 72 h did not committed the cells into apoptosis as poly(ADP-ribose) polymerase-1 (hPARP-1), a 113-kDa DNA repair enzyme whose cleavage by caspase-3 into degradation products of 89- and 22-kDa (of which the larger is efﬁciently recognized by the C-2–10 mAb) is recognized as an early marker of apop- tosis (52,53), remained entirely intact during serum starvation (Figure 6D). This is further validated by the fact that proteins in the high molecular range showed no evidence of degradation upon Coomassie blue staining on gel (Figure 6E). In addition, HSFs exhibited a normal cell morphology under phase-contrast microscopy with no sign of apoptosis, even when the cells were maintained for 72 h without serum (Figure 6F). NFI binds the p21 promoter in vivo in dividing but not serum-starved HSFs The binding of NFI to the p21 gene promoter area was further examined in vivo in HSFs by ChIP assays. As a control, ChIP was also conducted on other transcription factors, such as Sp1, Sp3 and E2F1, also reported to bind the p21 basal pro- moter. Primers that spanned the entire p21 promoter from 279 down to +223 relative to the p21 mRNA start site were selected for the assay. Antibodies against Sp1, Sp3 and NFI all enriched the p21 promoter sequences in HSFs grown in complete, serum-containing medium, indicating that this genomic area is bound in vivo by these transcrip- tion factors (Figure 7A). In contrast, only the Sp3 and E2F1 antibodies enriched the p21 promoter in HSFs main- tained under serum starvation for 72 h, relative to the ‘no antibody’ control sample. These results suggest that Sp1, Sp3 and NFI are bound to cis-acting elements present on the p21 promoter in the in vivo chromatin conﬁguration of HSF cells during the dividing phase of the cell cycle (basal level of p21). Upon serum starvation (where high levels of p21 expression are observed), binding of both Sp1 and NFI were completely abrogated. Interestingly, while the level of Sp3 binding to the p21 promoter remained unaff- ected by serum starvation, that of E2F1, initially undetectable in actively growing cells, was found to bind weakly in serum- deprived HSFs. The signiﬁcant occupancy of NFI in relation to the ‘input’ (Figure 7B), suggest a critical role for this transcription factor on the p21 gene regulation. These results demonstrate that NFI binds the p21 promoter area in vivo in actively growing, but not serum-starved, growth- arrested HSFs. Suppression of NFI expression by RNAi alleviates p21 repression Evidence that NFI functionally represses p21 promoter activ- ity was further examined through the suppression of the