DNA binding of NF-Y: the effect of HMGI proteins depends upon the CCAAT box

AbstractNF-Y is a conserved sequence-specific transcription factor binding to CCAAT boxes. The chromatin-associated HMGI proteins influence promoter activities through positive and negative effects on binding of transcription factors. It was previously shown that HMGI(Y) synergizes the binding of NF-Y to the α2-collagen CCAAT box [Currie, R.A. (1997) J. Biol Chem. 272, 30880–30888]. Using recombinant proteins, we confirm that at low concentrations of NF-Y, HMGI(Y) acts synergistically on the α2-collagen CCAAT and we extend this observation to HMGI and HMGI-C. However, enhancement of DNA binding to γ-globin, α-globin and MHC class II Ea CCAAT boxes was not observed. At high concentrations, HMGI proteins inhibit binding to α2-collagen and to γ-globin, but not to high affinity Ea or α-globin CCAAT. In none of our experiments did we see a ternary complex between NF-Y, HMGI(Y) and DNA. In protein competition experiments, NF-Y affinity was at least two orders of magnitude higher, even in the context of the suboptimal γ-globin CCAAT. Our data prove that HMGI proteins have complex positive and negative effects on NF binding to some, but not to all CCAAT boxes, suggesting that this phenomenon is dictated by the sequences flanking the pentanucleotide rather than direct protein-protein interactions