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• W2063063973 abstract "The high mobility group HMG I(Y) protein has been reported to promote the expression of several NF-κB-dependent genes by enhancing the binding of NF-κB to DNA. The molecular origins of cooperativity in the binding of NF-κB 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-κB, to two different DNA elements, PRDII from the interferon-β enhancer and IgκB from the immunoglobulin κ 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-κB to PRDII and IgκB 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-κB. 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-κB 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-κB to PRDII and IgκB elements. The high mobility group HMG I(Y) protein has been reported to promote the expression of several NF-κB-dependent genes by enhancing the binding of NF-κB to DNA. The molecular origins of cooperativity in the binding of NF-κB 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-κB, to two different DNA elements, PRDII from the interferon-β enhancer and IgκB from the immunoglobulin κ 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-κB to PRDII and IgκB 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-κB. 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-κB 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-κB to PRDII and IgκB elements. HMG 1The abbreviations HMGhigh mobility groupIfninterferonPRDpositive regulatory domainATFactivating transcription factorRHRRel homology regionGSTglutathione S-transferaseIREinterferon response elementbpbase pair(s)EMSAelectrophoretic mobility shift assayONPGo-nitrophenyl-β-d-galactopyranosideBSAbovine serum albuminADactivation domain 1The abbreviations HMGhigh mobility groupIfninterferonPRDpositive regulatory domainATFactivating transcription factorRHRRel homology regionGSTglutathione S-transferaseIREinterferon response elementbpbase pair(s)EMSAelectrophoretic mobility shift assayONPGo-nitrophenyl-β-d-galactopyranosideBSAbovine serum albuminADactivation domainI(Y) protein belongs to the family of high mobility group (HMG) non-histone chromosomal proteins that binds preferentially in the minor groove of AT-rich sequences in DNA (1Bustin M. Lehn D.A. Landsman D. Biochim. Biophys. Acta. 1990; 1049: 231-243Crossref PubMed Scopus (438) Google Scholar). The expression of HMG I(Y) is normally very low in adult tissues, but is up-regulated in the rapidly proliferating cells found in embryonic and neoplastic tissues, leading to the suggestion that the HMG I(Y) gene might be directly involved in development and neoplasia (2Johnson K.R. Lehn D.A. Elton T.S. Barr P.J. Reeves R. J. Biol. Chem. 1988; 263: 18338Abstract Full Text PDF PubMed Google Scholar). HMG I(Y) has also been implicated as a structural component involved in the condensation of AT-rich regions of mammalian chromosomes (3Johnson K.R. Lehn D.A. Reeves R. Mol. Cell. Biol. 1989; 9: 2114-2123Crossref PubMed Scopus (223) Google Scholar), and has been shown to be a host protein required for integration by HIV-1 preintegration complexes in vitro (4Farnet C.M. Bushman F.D. Cell. 1997; 88: 483-492Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar).HMG I and HMG Y are isoforms produced by alternative splicing of a single functional pre-mRNA, with 11 internal amino acids present in HMG I but deleted in HMG Y (3Johnson K.R. Lehn D.A. Reeves R. Mol. Cell. Biol. 1989; 9: 2114-2123Crossref PubMed Scopus (223) Google Scholar); the function of this 11-amino acid stretch is not known, hence the two isoforms are designated interchangeably as HMG I(Y). HMG I(Y) and HMG I-C, the other member of HMG I(Y) family, are both small (∼10 kDa) proteins, which share three highly conserved basic regions arranged in tandem, in addition to an acidic COOH-terminal domain. The basic regions are essential for DNA binding, whereas the COOH-terminal domain facilitates sequence-specific interaction with DNA (5Yie J. Liang S. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 3649-3662Crossref PubMed Scopus (107) Google Scholar). The consensus basic repeats possess a unique structure referred to as an “AT-hook,” which binds deeply into the minor groove of the AT-rich DNA recognition sites (6Huth J.R. Bewley C.A. Nissen M.S. Evans J.N. Reeves R. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 657-665Crossref PubMed Scopus (304) Google Scholar, 7Reeves R. Nissen M.S. J. Biol. Chem. 1990; 265: 8573-8582Abstract Full Text PDF PubMed Google Scholar).HMG I family proteins can serve as either positive or negative accessory factors for gene transcription regulated by a wide range of transcription factors, including those of the Rel, bZIP, ETS, and POU families (8Du W. Thanos D. Maniatis T. Cell. 1993; 74: 887-898Abstract Full Text PDF PubMed Scopus (395) Google Scholar, 9John S. Reeves R.B. Lin J.X. Child R. Leiden J.M. Thompson C.B. Leonard W.J. Mol. Cell. Biol. 1995; 15: 1786-1796Crossref PubMed Google Scholar, 10Leger H. Sock E. Renner K. Grummt F. Wegner M. Mol. Cell. Biol. 1995; 15: 3738-3747Crossref PubMed Scopus (92) Google Scholar, 11Thanos D. Maniatis T. Cell. 1992; 71: 777-789Abstract Full Text PDF PubMed Scopus (557) Google Scholar). HMG I proteins do not appear to function independently as transcriptional activators or repressors (12Landsman D. Bustin M. Mol. Cell. Biol. 1991; 11: 4483-4489Crossref PubMed Scopus (41) Google Scholar), even though they possess an acidic domain reminiscent of that often found in many transcriptional activator proteins (13Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1167) Google Scholar). Instead, they bind to certain AT-rich sequences within or flanking transcription factor recognition sites, either enhancing (10Leger H. Sock E. Renner K. Grummt F. Wegner M. Mol. Cell. Biol. 1995; 15: 3738-3747Crossref PubMed Scopus (92) Google Scholar, 11Thanos D. Maniatis T. Cell. 1992; 71: 777-789Abstract Full Text PDF PubMed Scopus (557) Google Scholar) or competing with (14Arlotta P. Rustighi A. Mantovani F. Manfioletti G. Giancotti V. Tell G. Damante G. J. Biol. Chem. 1997; 272: 29904-29910Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 15Klein-Hessling S. Schneider G. Heinfling A. Chuvpilo S. Serfling E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15311-15316Crossref PubMed Scopus (57) Google Scholar) the binding of these transcription factors to adjacent or overlapping DNA sites.Among the loci reported to be regulated by HMG I family proteins, the most extensively characterized is the human interferon-β (Ifn-β) gene (16Maniatis T. Falvo J.V. Kim T.H. Kim T.K. Lin C.H. Parekh B.S. Wathelet M.G. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 609-620Crossref PubMed Scopus (315) Google Scholar), which is transcriptionally activated upon viral infection. The gene enhancer consists of a negative regulatory domain NRDI (17Nourbakhsh M. Hoffmann K. Hauser H. EMBO J. 1993; 12: 451-459Crossref PubMed Scopus (58) Google Scholar), as well as four adjacent positive regulatory domains (PRDs) organized in the order: PRDII, PRDI-PRDIII, and PRDIV; these are recognized, respectively, by the transcription factors NF-κB (18Visvanathan K.V. Goodbourn S. EMBO J. 1989; 8: 1129-1138Crossref PubMed Scopus (213) Google Scholar), Ifn-regulatory factor 1 (19Fujita T. Kimura Y. Miyamoto M. Barsoumian E.L. Taniguchi T. Nature. 1989; 337: 270-272Crossref PubMed Scopus (315) Google Scholar), and activating transcription factor 2 (ATF-2)/c-Jun (20Du W. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2150Crossref PubMed Scopus (96) Google Scholar). HMG I(Y) binds specifically to one site within NRDI, one site in PRDII, and two sites in PRDIV (5Yie J. Liang S. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 3649-3662Crossref PubMed Scopus (107) Google Scholar). Rather than four HMG I(Y) molecules individually contacting these four different sites, recent evidence indicate that two HMG I(Y) molecules bind the enhancer, with each protein molecule occupying two closely juxtaposed sites. Specifically, the second and third repeats of HMG I(Y) simultaneously bind PRDII and NRDI, whereas the second and first repeats bind the two sites within PRDIV (5Yie J. Liang S. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 3649-3662Crossref PubMed Scopus (107) Google Scholar, 21Maher J.F. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6716-6720Crossref PubMed Scopus (144) Google Scholar). In both cases, the tandem HMG I(Y)-binding sites are separated by one full helical turn, and the binding interaction is highly cooperative. All four HMG I(Y) sites within the enhancer are essential for optimal virus induction of the Ifn-β gene. The binding of HMG I(Y) to the minor groove of PRDII stimulates NF-κB binding to the major groove of the same site approximately 10-fold in vitro (11Thanos D. Maniatis T. Cell. 1992; 71: 777-789Abstract Full Text PDF PubMed Scopus (557) Google Scholar). Similarly, the affinity of ATF-2 for PRDIV increases about 4-fold upon coincubation with HMG I(Y) (8Du W. Thanos D. Maniatis T. Cell. 1993; 74: 887-898Abstract Full Text PDF PubMed Scopus (395) Google Scholar). Circular permutation and phasing analyses of Ifn-β gene enhancer revealed that the intrinsic DNA bends in the enhancer can be reversed by the binding of NF-κB and ATF-2/c-Jun, and further modulated by HMG I(Y) binding (22Falvo J.V. Thanos D. Maniatis T. Cell. 1995; 83: 1101Abstract Full Text PDF PubMed Scopus (276) Google Scholar). Taken together, these studies establish an important role for HMG I(Y) as an accessory protein which mediates the assembly and function of the Ifn-β gene enhanceosome by promoting the cooperative binding of transcription factors to the enhancer (23Thanos D. Maniatis T. Cell. 1995; 83: 1091-1100Abstract Full Text PDF PubMed Scopus (847) Google Scholar, 24Kim T.K. Maniatis T. Mol. Cell. 1998; 1: 119-129Abstract Full Text Full Text PDF Scopus (298) Google Scholar).NF-κB is the prototype of the dimeric class of Rel proteins, members of which are characterized by the presence of an approximately 300-amino acid Rel homology region (RHR) that mediates protein-protein interactions, sequence-specific DNA recognition, and nuclear translocation (25Chytil M. Verdine G.L. Curr. Opin. Struct. Biol. 1996; 6: 91-100Crossref PubMed Scopus (42) Google Scholar). NF-κB plays a broad role in inducible and coordinate regulation of genes involved in inflammation, immune responses, cell growth and differentiation, and virus infection (26Baeuerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2916) Google Scholar). Evidence suggests that NF-κB cooperates with HMG I(Y) at a variety of regulatory loci. In addition to the Ifn-β gene, HMG I(Y) has also been reported to enhance NF-κB activity on several other promoters including E-selectin (27Lewis H. Kaszubska W. DeLamarter J.F. Whelan J. Mol. Cell. Biol. 1994; 14: 5701-5709Crossref PubMed Google Scholar), interleukin-2/granulocyte-macrophate colony-stimulating factor (28Himes S.R. Coles L.S. Reeves R. Shannon M.F. Immunity. 1996; 5: 479-489Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), and chemokine MGSA/GRO α (29Wood L.D. Farmer A.A. Richmond A. Nucleic Acids Res. 1995; 23: 4210-4219Crossref PubMed Scopus (80) Google Scholar).Despite the importance of the cooperative interactions between NF-κB and HMG I(Y), the mechanistic basis for this phenomenon is poorly understood. In principle, cooperative binding of NF-κB and HMG I(Y) to DNA could arise from either or both of two limiting scenarios: (i) the binding of one protein to its site might induce local structure changes in DNA that favor interaction of the other protein with its site; or (ii) NF-κB and HMG I(Y) might make favorable protein-protein contacts with each other. Consistent with the first scenario, HMG I(Y) can reduce the intrinsic DNA curvature of PRDII and enhance the reversal of PRDII bending by NF-κB (22Falvo J.V. Thanos D. Maniatis T. Cell. 1995; 83: 1101Abstract Full Text PDF PubMed Scopus (276) Google Scholar). In keeping with the second scenario, HMG I(Y) has been shown to bind both NF-κB and ATF-2 in GST fusion protein binding assays (8Du W. Thanos D. Maniatis T. Cell. 1993; 74: 887-898Abstract Full Text PDF PubMed Scopus (395) Google Scholar, 9John S. Reeves R.B. Lin J.X. Child R. Leiden J.M. Thompson C.B. Leonard W.J. Mol. Cell. Biol. 1995; 15: 1786-1796Crossref PubMed Google Scholar, 10Leger H. Sock E. Renner K. Grummt F. Wegner M. Mol. Cell. Biol. 1995; 15: 3738-3747Crossref PubMed Scopus (92) Google Scholar), a procedure that can sometimes detect weak interactions that do not occur physiologically. Direct evidence has not been provided to show that NF-κB and HMG I(Y) can make energetically favorable contacts to each other on DNA recognition elements in vivo. Therefore, the contribution of direct protein-protein interactions to the cooperative binding of NF-κB and HMG I(Y) to DNA remains uncertain.To investigate this issue and further analyze the NF-κB/HMG I(Y)/DNA interaction interface, while avoiding the potential complexity arising from the abundant endogenous HMG I(Y) and NF-κB proteins in mammalian cells, we have reconstituted the core NF-κB·HMG I·DNA complex in yeast. Using this system, we investigated the DNA binding specificity of HMG I(Y) on two κB DNA motifs (the core NF-κB sites from PRDII and IgκB). We demonstrate that the discrimination of these two κB sites by HMG I(Y), in terms of both DNA recognition and cooperation with NF-κB, can be greatly enhanced by the presence of additional AT-rich sequences adjacent to the core κB DNA motifs. The use of yeast one-hybrid screening assays (30Peterson B.R. Sun L.J. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13671-13676Crossref PubMed Scopus (51) Google Scholar, 31Sun L.J. Peterson B.R. Verdine G.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4919-4924Crossref PubMed Scopus (42) Google Scholar) in combination with deletion and alanine-scanning mutagenesis (32Cunningham B.C. Wells J.A. Science. 1989; 244: 1081-1085Crossref PubMed Scopus (1078) Google Scholar) of HMG I enabled us to examine and compare mutations affecting NF-κB/HMG I cooperativity and HMG I/DNA recognition on the PRDII or IgκB site, which either alone or flanked by NRDI as in the context of the entire interferon response element (IRE, including PRDI, PRDII, and NRDI). Intriguingly, we find that the mutations affecting NF-κB/HMG I cooperative DNA binding also affect HMG I DNA affinity to a similar extent. While the residues most important for core NF-κB site recognition fall within the second basic repeat of HMG I, the third repeat is identified as being crucial for HMG I/PRDII-NRDI interaction. Further in vitrocharacterization of the protein-protein interaction independent of DNA revealed that the same mutations in the second repeat abolished the interaction of HMG I with NF-κB p50 as well. Therefore, binding sites for the same region of HMG I exist in both NF-κB p50 protein and κB DNA motifs. These data imply that the second repeat of HMG I, through either binding to κB DNA motifs, or interacting with NF-κB p50, or both, contributes the most to the cooperative binding of NF-κB and HMG I to DNA.DISCUSSIONThe enhanceosome model for transcriptional activation emphasizes the importance of cooperative interactions among regulatory proteins and the transcriptional apparatus as a means of stabilizing an initiation-competent complex (23Thanos D. Maniatis T. Cell. 1995; 83: 1091-1100Abstract Full Text PDF PubMed Scopus (847) Google Scholar, 24Kim T.K. Maniatis T. Mol. Cell. 1998; 1: 119-129Abstract Full Text Full Text PDF Scopus (298) Google Scholar). HMG I(Y) is unique in several respects among cooperative partners: (i) the protein is exceptionally broad in its DNA sequence preferences; (ii) HMG I(Y) contains no intrinsic transcriptional activation domain, and most probably acts only to enhance the affinity of its partner protein for DNA; and (iii) HMG I(Y) cooperates with an unusually diverse array of structurally unrelated proteins, including NF-κB, ATF-2/c-Jun, Tst-1, and Elf-1 (8Du W. Thanos D. Maniatis T. Cell. 1993; 74: 887-898Abstract Full Text PDF PubMed Scopus (395) Google Scholar, 9John S. Reeves R.B. Lin J.X. Child R. Leiden J.M. Thompson C.B. Leonard W.J. Mol. Cell. Biol. 1995; 15: 1786-1796Crossref PubMed Google Scholar, 10Leger H. Sock E. Renner K. Grummt F. Wegner M. Mol. Cell. Biol. 1995; 15: 3738-3747Crossref PubMed Scopus (92) Google Scholar, 11Thanos D. Maniatis T. Cell. 1992; 71: 777-789Abstract Full Text PDF PubMed Scopus (557) Google Scholar). A major challenge is to understand how HMG I exerts such specific effects on transcription while exhibiting such apparent promiscuity in binding. The present studies were aimed at gaining insight into these issues through in vitro and in vivo analyses of HMG I and one of its cooperative partners, NF-κB.An intriguing clue to the mystery of specificity in HMG I/DNA interactions was gained by the observation that the tandem repeats in HMG I can interact simultaneously with appropriately phased AT-rich tracts in the interferon-β promoter (5Yie J. Liang S. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 3649-3662Crossref PubMed Scopus (107) Google Scholar). This intramolecularly cooperative binding mode was found to enhance the affinity of HMG I/DNA interactions, thereby raising the possibility that specificity might also be affected. Indeed, our results confirm the fact that intramolecular cooperativity substantially strengthens HMG I/DNA interactions; furthermore, we find that the specificity of HMG I for tandem AT-rich sites is significantly increased over that for single sites alone. This dichotomy in DNA-binding mode by HMG I may help to explain the weak discrimination for the core PRDII versusIgκB sites observed in our studies, while others have reported stronger discrimination. A critical difference between these studies lies in the fact that we purposely excluded AT-rich flanking sequences from our core κB probes, whereas the naturally occurring DNA elements used previously contained such AT-rich flanking elements as a natural consequence of proper binding function. Remarkably, HMG I shows less than 5-fold discrimination for DNA containing a single AT-rich tract (PRDII) over mixed sequence DNA containing no AT-tract, but increases to nearly 20-fold when the AT-rich tract is linked to another in NRDI.The effect of tandem intramolecular binding by HMG I on its cooperation with NF-κB has not been examined previously. Here we find that the binding of HMG I to either the core PRDII or IgκB site does little if anything to stimulate NF-κB binding (Fig. 4 B). By contrast, HMG I powerfully stimulates the binding of NF-κB to the tandemly arrayed PRDII-NRDI site (Fig. 4 A). This stimulation does not appear to result simply from tandem intramolecular binding of HMG I, but rather in some more complex way from the DNA sequence, as little cooperativity between HMG I and NF-κB was evident on the IgκB-NRDI site (Fig. 4 A).What is the mechanism of cooperative DNA binding by HMG I and NF-κB on PRDII-NRDI? The near perfect correlation between the effects of mutations on reporter assays driven by HMG I alone and in the presence of NF-κB is most consistent with an indirect readout mechanism, wherein the binding of HMG I alters DNA structure so as to make it more favorable for NF-κB recognition. Indeed, the PRDII site contains an intrinsic bend, which is reversed upon NF-κB binding (22Falvo J.V. Thanos D. Maniatis T. Cell. 1995; 83: 1101Abstract Full Text PDF PubMed Scopus (276) Google Scholar); perhaps HMG I lowers the energetic cost of this reversal. Paradoxically, however, several alanine mutations that disturb HMG I-DNA binding and HMG I/NF-κB cooperation also weaken protein-protein interactions between HMG I and NF-κB (Fig. 7), suggesting the two proteins might cooperate through direct contacts as well. This paradox could be resolved if the very same residues in the second basic repeat of HMG I that contact DNA also contact NF-κB. As no experimental data are presently available to address this question, nor was a simple, direct and definitive experimental test immediately apparent, we turned to the NMR structure of the HMG I (51–90) fragment bound to DNA (6Huth J.R. Bewley C.A. Nissen M.S. Evans J.N. Reeves R. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 657-665Crossref PubMed Scopus (304) Google Scholar). As revealed by this structure, a central Arg-Gly-Arg core (residues 58–60) in the second repeat of HMG I adopts an extended conformation and penetrates deep into the DNA minor groove. Importantly, NF-κB is bound exclusively in the major groove on the backside of DNA. From the perspective of this NMR structure, the spatial separation between the HMG I- and NF-κB-binding sites renders it exceedingly difficult, if not impossible, for multiple DNA contact residues of the HMG I second basic repeat to simultaneously contact residues of NF-κB. Either of two possibilities seems more likely: (i) protein-protein contacts between HMG I and NF-κB that are responsible for GST pull-down result from some weak interaction between the two proteins that bears no relevance to cooperative complex formation; or (ii) perhaps the overall structure of HMG I changes substantially with respect to DNA in going from the binary to ternary complexes, thereby allowing the protein to interact with both DNA and NF-κB using the second basic repeat.If NF-κB and HMG I cooperate by making mutually favorable alterations of DNA structure, then it is possible to rationalize straightforwardly the marked sequence dependence of cooperativity observed in our in vitro cooperativity assays (Fig.4 A). Specifically, binding of HMG I or NF-κB to one site may induce a DNA structure that favors binding of the other protein, whereas a different DNA sequence cannot adopt the same structure. It is noteworthy that one of the two base pairs that differ in the PRDII and IgκB sites is contacted directly by the p50 subunit of NF-κB: K244 of p50 hydrogen bonds to the T residue of the 5′-GGGAAATTCC-3′·5′-GGAATTTCCC-3′ (PRDII) or to the corresponding G residue in IgκB. Likewise, the NMR structure of HMG I peptide bound to DNA reveals that residues Arg-58, Gly-59, and Arg-60 (second repeat) or Arg-84, Gly-85, and Arg-86 (third repeat) make extensive minor groove contacts to both base pairs that differ in PRDII and IgκB (6Huth J.R. Bewley C.A. Nissen M.S. Evans J.N. Reeves R. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 657-665Crossref PubMed Scopus (304) Google Scholar). The fact that both partners contact the same base pairs in PRDII and IgκB, although from different grooves, provides a clear mechanism for the two partners to communicate with one another through DNA. It stands to reason that the ability to communicate and thus bind DNA cooperatively should depend upon the covalent structure and conformation of the bound DNA sequence.The inability to pinpoint precisely the mechanism of cooperativity between HMG I and NF-κB reflects in no small part the inherent difficulty of studying proteins such as HMG I that exhibit relatively weak specificity, and possess little or no secondary or tertiary structure, even when bound to DNA. There exists a great need for new experimental approaches to study such problems. It is worth mentioning that even alanine-scanning mutagenesis fails to detect any potentially important interactions involving the polypeptide backbone and α-carbons, hence important information on cooperative binding may not be revealed with this technique.Three highly conserved basic regions in HMG I(Y) have been shown to be responsible for the specific recognition of AT-rich sequences (7Reeves R. Nissen M.S. J. Biol. Chem. 1990; 265: 8573-8582Abstract Full Text PDF PubMed Google Scholar). Interestingly, multiple copies of this motif are also found in several eukaryotic proteins that bind AT-rich sequences and whose genes are involved in chromosome translocations associated with human disease (44Tjaden G. Coruzzi G.M. Plant Cell. 1994; 6: 107-118PubMed Google Scholar, 45Tkachuk D.C. Kohler S. Cleary M.L. Cell. 1992; 71: 691-700Abstract Full Text PDF PubMed Scopus (856) Google Scholar). The interaction modes of the second and the third repeats with DNA have been illustrated in the HMG I (51–90)/DNA NMR structure (6Huth J.R. Bewley C.A. Nissen M.S. Evans J.N. Reeves R. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 657-665Crossref PubMed Scopus (304) Google Scholar). In our study, we mutated residues in each basic repeat to alanines in the context of full-length HMG I and then assayed the binding activity of each mutant in vivo and in vitro. On a single AT-rich binding site, the second repeat seems to make the greatest energetic contribution to the overall protein-DNA interaction, as judged by the strong effect of mutations in this region. Nevertheless, high-affinity binding of HMG I can be achieved through simultaneous interactions of multiple basic motifs with multiple AT tracts located in close proximity to each other (21Maher J.F. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6716-6720Crossref PubMed Scopus (144) Google Scholar). This multivalent HMG I DNA binding was indeed the case on the Ifn-β gene enhancer: simultaneous contacts were observed with either the second and third repeats to PRDII and NRDI within IRE, or the second and first repeats to two sites within PRDIV (5Yie J. Liang S. Merika M. Thanos D. Mol. Cell. Biol. 1997; 17: 3649-3662Crossref PubMed Scopus (107) Google Scholar). Being the most specific DNA-binding region in HMG I, the second repeat also shows the highest DNA binding affinity, being 10–100-fold greater than those of the other two repeats (6Huth J.R. Bewley C.A. Nissen M.S. Evans J.N. Reeves R. Gronenborn A.M. Clore G.M. Nat. Struct. Biol. 1997; 4: 657-665Crossref PubMed Scopus (304) Google Scholar). Together, these data indicate that the second repeat is the most important region for HMG I/DNA binding. The molecular details of inter- and intra-molecularly cooperative binding by HMG I await further investigation. HMG 1The abbreviations HMGhigh mobility groupIfninterferonPRDpositive regulatory domainATFactivating transcription factorRHRRel homology regionGSTglutathione S-transferaseIREinterferon response elementbpbase pair(s)EMSAelectrophoretic mobility shift assayONPGo-nitrophenyl-β-d-galactopyranosideBSAbovine serum albuminADactivation domain 1The abbreviations HMGhigh mobility groupIfninterferonPRDpositive regulatory domainATFactivating transcription factorRHRRel homology regionGSTglutathione S-transferaseIREinterferon response elementbpbase pair(s)EMSAelectrophoretic mobility shift assayONPGo-nitrophenyl-β-d-galactopyranosideBSAbovine serum albuminADactivation domainI(Y) protein belongs to the family of high mobility group (HMG) non-histone chromosomal proteins that binds preferentially in the minor groove of AT-rich sequences in DNA (1Bustin M. Lehn D.A. Landsman D. Biochim. Biophys. Acta. 1990; 1049: 231-243Crossref PubMed Scopus (438) Google Scholar). The expression of HMG I(Y) is normally very low in adult tissues, but is up-regulated in the rapidly proliferating cells found in embryonic and neoplastic tissues, leading to the suggestion that the HMG I(Y) gene might be directly involved in development and neoplasia (2Johnson K.R. Lehn D.A. Elton T.S. Barr P.J. Reeves R. J. Biol. Chem. 1988; 263: 18338Abstract Full Text PDF PubMed Google Scholar). HMG I(Y) has also been implicated as a structural component involved in the condensation of AT-rich regions of mammalian chromosomes (3Johnson K.R. Lehn D.A. Reeves R. Mol. Cell. Biol. 1989; 9: 2114-2123Crossref PubMed Scopus (223) Google Scholar), and has been shown to be a host protein required for integration by HIV-1 preintegration complexes in vitro (4Farnet C.M. Bushman F.D. Cell. 1997; 88: 483-492Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). high mobility group interferon positive regulatory domain activating transcription factor Rel homology region glutathione S-transferase interferon response element base pair(s) electrophoretic mobility shift assay o-nitrophenyl-β-d-galactopyranoside bovine serum albumin activation dom" @default.
• W2063063973 created "2016-06-24" @default.
• W2063063973 creator A5005061888 @default.
• W2063063973 creator A5034429970 @default.
• W2063063973 date "1999-07-01" @default.
• W2063063973 modified "2023-10-17" @default.
• W2063063973 title "A Small Region in HMG I(Y) Is Critical for Cooperation with NF-κB on DNA" @default.
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