A small region in HMGI I(Y) is critical for cooperation with NF-κB on DNA

The high mobility group HMG I(Y) protein has been reported to promote the expression of several NF-kappa B-dependent genes by enhancing the binding of NF-kappa B to DNA. The molecular origins of cooperativity in the binding of NF-kappa B and HMG I(Y) to DNA are not well understood. Here we have examined the determinants of specificity in the binding of HMG I(Y), both alone and in cooperation with NF-kappa B, to two different DNA elements, PRDII from the interferon-beta enhancer and Ig kappa B from the immunoglobulin kappa light chain enhancer. Of particular interest was the influence of a flanking AT-rich sequence on binding by HMG I(Y). Utilizing yeast one-hybrid screening assays together with alanine-scanning mutagenesis, we have identified mutations of residues in HMG: I(Y) that decrease cooperative binding of NF-kappa B to PRDII and Ig kappa B sites. These same mutations similarly decreased the binding of HMG: I(Y) alone to DNA, and paradoxically, decreased the strength of protein-protein interactions between HMG I(Y) and NF-kappa B. Of the three tandemly repeated basic regions that represent putative DNA-binding motifs in HMG; I(Y), the residues within the second repeat are most important for recognition of core NF-kappa B sites, whereas the second and third repeats both appear to be involved in binding to sites that are flanked by AT-rich sequences. Overall, the second repeat of HMG I(Y) is primarily responsible for the stimulatory effect of this protein on the binding of NF-kappa B to PRDII and Ig kappa B elements.