Figure 3. Dietary PUFAs impair NF-Y DNA binding activity.

Hepatic nuclear protein extracts (5 μg per reaction) were prepared from rats fed an FF diet or the FO diet. (A) EMSAs were conducted using the Fasn promoter region of −106 to −77 bp in which the candidate Sp1 site was mutated (−106/−77_Sp1mt) (see Table 1). Lane 1 is free probe. Lanes 2 and 3 show NF-Y binding activity in extracts from rats fed the FF diet or the FO diet respectively. Specificity of NF-Y binding to the −106/−77_Sp1mt fragment (A) was determined using nuclear proteins from rats fed the FF diet incubated with a 100-fold molar excess of unlabelled −106/−77_Sp1mt (lane 4), NF-Y consensus sequence (lane 5), wild-type −106 to −77 bp Fasn sequence (lane 6), or the anti-(NF-YA) antibody (lane 7). (B) EMSAs were conducted using an oligonucleotide containing a consensus NF-Y binding sequence (see Table 1) and nuclear protein extracts from rats fed the FF diet (lane 1) or the FO diet (lane 2). Specificity of NF-Y binding to the consensus sequence was determined using nuclear proteins from rats fed the FF diet incubated with a 100-fold molar excess of unlabelled consensus NF-Y site (lane 3) or the anti-(NF-YA) antibody (lane 4). The left-hand arrows indicate the NF-Y binding complex, and the right-hand arrow indicates the super-shift resulting from treatment with anti-(NF-YA) antibody. Reduced NF-Y binding by dietary FO was demonstrated in four rats with a mean decrease in binding activity of 50±7% (P<0.05) for the −106/−77_Sp1mt and consensus NF-Y sequences.