A Small Region in HMG I(Y) Is Critical for Cooperation with NF-κB on DNA

• Published in: The Journal of biological chemistry Vol. 274; no. 29; pp. 20235 - 20243
• Main Authors: Zhang, Xiaoyan Michelle, Verdine, Gregory L.
• Format: Journal Article
• Language: English
• Published: 16.07.1999
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.29.20235

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.