Zhang X M, Verdine G L
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
J Biol Chem. 1999 Jul 16;274(29):20235-43. 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-kappaB-dependent genes by enhancing the binding of NF-kappaB to DNA. The molecular origins of cooperativity in the binding of NF-kappaB 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-kappaB, to two different DNA elements, PRDII from the interferon-beta enhancer and IgkappaB 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-kappaB to PRDII and IgkappaB 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-kappaB. 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-kappaB 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-kappaB to PRDII and IgkappaB elements.
据报道，高迁移率族蛋白HMG I(Y)可通过增强NF-κB与DNA的结合来促进多种NF-κB依赖性基因的表达。目前对于NF-κB和HMG I(Y)协同结合DNA的分子机制还了解甚少。在此，我们研究了HMG I(Y)单独结合以及与NF-κB协同结合两种不同DNA元件（来自干扰素-β增强子的PRDII和来自免疫球蛋白κ轻链增强子的IgκB）时的特异性决定因素。特别值得关注的是富含AT的侧翼序列对HMG I(Y)结合的影响。利用酵母单杂交筛选试验和丙氨酸扫描诱变，我们鉴定出了HMG I(Y)中一些残基的突变，这些突变降低了NF-κB与PRDII和IgκB位点的协同结合。同样，这些突变也降低了HMG I(Y)单独与DNA的结合，而且矛盾的是，还降低了HMG I(Y)与NF-κB之间蛋白质-蛋白质相互作用的强度。在HMG I(Y)中代表假定DNA结合基序的三个串联重复的碱性区域中，第二个重复区域内的残基对于识别核心NF-κB位点最为重要，而第二个和第三个重复区域似乎都参与了与富含AT序列侧翼位点的结合。总体而言，HMG I(Y)的第二个重复区域主要负责该蛋白对NF-κB与PRDII和IgκB元件结合的刺激作用。
