Intra- and Intermolecular Cooperative Binding of High-Mobility- Group ...

MOLECULAR AND CELLULAR BIOLOGY, July 1997, p. 3649–3662 0270-7306/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 17, No. 7

Intra- and Intermolecular Cooperative Binding of High-MobilityGroup Protein I(Y) to the Beta-Interferon Promoter JUNMING YIE, STANLEY LIANG, MENIE MERIKA,

AND

DIMITRIS THANOS*

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 Received 3 March 1997/Returned for modification 31 March 1997/Accepted 9 April 1997

The mammalian high-mobility-group protein I(Y) [HMG I(Y)], while not a typical transcriptional activator, is required for the expression of many eukaryotic genes. HMG I(Y) appears to recruit and stabilize complexes of transcriptional activators through protein-DNA and protein-protein interactions. The protein binds to the minor groove of DNA via three short basic repeats, preferring tracts of adenines and thymines arranged on the same face of the DNA helix. However, the mode by which these three basic repeats function together to recognize HMG I(Y) binding sites has remained unclear. Here, using deletion mutants of HMG I(Y), DNase I footprinting, methylation interference, and in vivo transcriptional assays, we have characterized the binding of HMG I(Y) to the model beta-interferon enhancer. We show that two molecules of HMG I(Y) bind to the enhancer in a highly cooperative fashion, each molecule using a distinct pair of basic repeats to recognize the tandem AT-rich regions of the binding sites. We have also characterized the function of each basic repeat, showing that only the central repeat accounts for specific DNA binding and that the presence of a second repeat bound to an adjacent AT-rich region results in intramolecular cooperativity in binding. Surprisingly, the carboxyl-terminal acidic tail of HMG I(Y) is also important for specific binding in the context of the full-length protein. Our results present a detailed examination of HMG I(Y) binding in an important biological context, which can be extended not only to HMG I(Y) binding in other systems but also to the binding mode of many other proteins containing homologous basic repeats, which have been conserved from bacteria to humans. cluding Rel (37), bZip (9), Ets (20), and POU (23), typically stabilizing their binding to DNA. An increasing number of cytokine and viral genes have been shown to be regulated by HMG I(Y). These include genes for cytokines such as beta interferon (IFN-b) (9, 34), E-selectin (24, 27, 43), class II DRA (1), interleukin 2 (IL-2) receptor a chain (20), granulocytemacrophage colony-stimulating factor and IL-2 (19), MGSA/ GROa (46), human papilloma virus JC product (23), and the Epstein-Barr virus EBNA1 gene (12). Other studies have also shown that HMG I(Y) antagonizes histone H1-mediated repression and that it plays a critical role in chromosomal architecture (36, 47). The HMG I(Y) and HMG I-C genes are rearranged in a variety of benign tumors, and a null mutation of the HMG I-C gene in mice decreases the rate of cell proliferation, resulting in the pygmy phenotype (2, 34, 48). More recently, HMG I(Y) was shown to be important for the function of human immunodeficiency virus type 1 preintegration complexes in vitro (11). Clearly, HMG I(Y) proteins are involved in many important biological processes. HMG I(Y) is an architectural factor (45) essential for the highly specific and synergistic virus induction of the human IFN-b gene recruiting the transcriptional activators NF-kB, ATF-2/c-Jun, and IRF-1 to the PRDII, PRDIV, and PRDIII-I elements of the enhancer (8, 9, 37–39). HMG I(Y) alters the conformation of the DNA (10) and facilitates the assembly of an enhanceosome, a higher-order nucleoprotein complex containing at least the three types of transcription factors listed above and HMG I(Y) (39). Although previously described architectural proteins such as integration host factor and lymphoid enhancer factor 1 facilitate interactions between proteins bound to widely separated sites by inducing sharp bends in DNA (reviewed in references 14, 16, and 17), HMG I(Y) induces modest changes in DNA structure (10) and binds to multiple sites closely linked to or contained within those of transcription factors, a pattern observed for several gene pro-

The high-mobility-group (HMG) family of proteins were initially defined as the abundant heterogeneous nuclear nonhistone components of chromatin. The HMG proteins have a low molecular weight, are highly charged, and are acid soluble. This heterogeneous class of nuclear proteins has been classified into three distinct families: the HMG-1/2, HMG I(Y), and HMG 14/17 families (reviewed in references 5, 16, and 17). The HMG I family includes HMG I, HMG Y, and HMG I-C. The HMG I and HMG Y polypeptides [HMG I(Y) protein] are encoded by the same gene and are generated through alternative RNA splicing (21), whereas the HMG I-C protein is encoded by a separate gene (26). Members of the HMG I family are distinguished from other groups of HMG proteins by their ability to bind specifically in the narrow minor groove of AT-rich DNA (reviewed in reference 6). The specific binding to DNA is mediated by three short basic repeats with a core motif of GRGRP or PRGRP (33). Nuclear magnetic resonance studies of the central repeat of HMG I (13), which contains the core motif PRGRP, verified the results of previous biochemical experiments (37) which indicated that HMG I contacts DNA from the minor groove. Interestingly, similar short basic repeats have been found in other proteins from bacteria (28), yeast, plants, Drosophila, and mammals. Examples include human HRX (ALL) (18, 41), Drosophila HMG D1 (3), chironomous cHMG I (7), pea ATBP-1 (32), rice PF1 (29), and yeast MIF2 and datin (4, 44). HMG I(Y) does not function as a transcriptional activator on its own but rather influences the activities of other regulatory factors (reviewed in reference 38). HMG I(Y) can interact directly with transcriptional activators of several families, in-

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th St., New York, NY 10032. Phone: (212) 305-6602. Fax: (212) 305-7932. 3649

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FIG. 1. HMG I(Y) binds cooperatively to the IFN-b promoter. Shown are results of a quantitative DNase I footprinting experiment using increasing amounts of recombinant HMG I and probes containing either the wild-type (WT) IFN-b promoter (A) or the following mutations: a mutation in the 59 HMG I site at PRDIV (B), a mutation in the 39 HMG I site at PRDIV (C), conversion of the PRDII element to the IgkB site (D), and mutation of the HMG I site at NRDI (E). In the wild-type promoter, HMG I binds simultaneously to five distinct sites (thick lines on the left side of the gel). In contrast, mutations in any of these sites result in decreased binding to the entire IFN-b promoter. The amounts of HMG I used were 30, 60, 120, 250, 500, and 1,000 ng. The slightly different pattern of DNase I cleavage obtained with the PRDIV-39 template is due to the addition of a BamHI restriction site upstream of the TATA box and to different polylinker sequences upstream of position 2110 (9). The corresponding HMG I binding sites in this template are indicated (thin lines on the left of the gel). The HMG I binding observed upstream of the PRDIV-59 mutation (B) is due to an additional HMG I site present between positions 2104 and 2110.

moters. Understanding of the molecular basis of HMG I(Y) binding to natural enhancers and promoters is critical because this binding likely represents the first step in the cooperative assembly of functional enhanceosomes (39) in numerous biological systems. In this study, we investigated the mechanisms by which HMG I(Y) binds to the IFN-b enhancer, using a panel of HMG I deletion mutants, DNase I footprinting, and methylation interference assays of wild-type and mutant enhancer sequences and transient transfections in HeLa and Drosophila S2 cells. We show that two pairs of AT-rich sequences positioned in phase on the DNA helix are required for highly cooperative binding of two molecules of HMG I(Y) to the enhancer. One molecule of HMG I(Y) binds to PRDII and the other binds to PRDIV in vitro, and both are required for enhancer function in vivo. We present a detailed structurefunction map for HMG I(Y) binding, demonstrating that the central basic repeat mediates specific DNA binding and cooperates with the first or third repeat for binding to PRDIV or PRDII, respectively. The first or third repeat on its own, however, fails to bind to DNA specifically. Finally, we show that in the context of the full-length protein, the acidic tail is required

for specific binding in vitro and enhancement of transcription in vivo. MATERIALS AND METHODS Cell culture and transfections. HeLa cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, antibiotics, and L-glutamine (2 mM) in a humidified incubator containing 5% CO2. One day prior to transfection, the cells were split in six-well plates (250,000 cells/well, 50 to 60% confluent), and the next day the cells were transfected by the calcium phosphate method. Two hours before transfection, the cells were refed with fresh media. The transfection cocktail contained 6 mg of reporter plasmid, 2 mg of cytomegalovirus–b-galactosidase expression vector, and 12 mg of pSP73 or pCDNA3 carrier plasmid. The day after transfection, the cells were washed four times with phosphate-buffered saline and infected with Sendai virus as described elsewhere (37). Eighteen hours later, the cells were harvested and the relative chloramphenicol acetyltransferase (CAT) activity was determined as previously described (37). Drosophila melanogaster SL2 cells were maintained in Schneider media (GIBCO BRL) supplemented with 12% heat-inactivated fetal calf serum and antibiotics at ambient temperature. One day prior to transfection, SL2 cells were plated in 12-well plates (106 cells/well) and transfected by the calcium phosphate method. The transfection cocktail contained 100 ng of reporter plasmid, 50 ng of Hsp82–b-galactosidase expression vector, and the amounts of activators indicated below. Vector DNA (pPAC) was added as necessary to achieve a constant amount of transfected DNA (3 mg).

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FIG. 2. (A) Diagrammatic representation of the HMG I deletions used in this study. Shown at the top is the maximum alignment of the amino acid sequences of HMG I, HMG Y, and HMG I-C. The numbering corresponds to HMG I. The three basic repeats are indicated (underlined). DNA binding reflects the ability of a derivative to bind DNA, whereas specific DNA binding reflects the ability to recognize specifically a DNA sequence as defined by DNase I footprinting and competitions in EMSAs. 1/2, low DNA binding activity (reduced more than 10-fold); (B) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel containing the indicated GST-fused HMG I deletion mutants after they have been purified on glutathione-agarose bead columns.

Plasmid constructions. The wild type and the PRDIV-59, PRDIV-39, and PII3kB mutations have been previously described (8, 9, 37, 39). Insertion and substitution mutations of the IFN-b promoter were constructed by using a PCR method described previously (37), and all mutations were verified by DNA sequencing. To generate the insertion of 5 and 10 bp between PRDII and the NRDI HMG I binding sites, the oligonucleotides GTGAAAGTGGGAAATTC CTN(5,10)CTGAATAGAGAGG (underlined are the HMG I binding sites) were used as primers in PCRs along with a CAT primer. A second PCR was performed using the CCCACTTTCACTTCTCCC oligonucleotide and T7 primer. The two PCR products were purified, annealed, and used in a new round of PCR with CAT and T7 primers. The final PCR product was restricted with XbaI and ClaI and cloned in pSP73-CAT (37). Two independent clones with different inserted sequences were selected for each mutation and used in the transfection experiments. The NRDI substitution mutation was generated similarly by using

the mutagenic oligonucleotide TGGGAAATTCCTCTtccgctcGAGAGGACCA. IFN-b enhancers containing double or triple mutations were generated as above by using singly or doubly mutated enhancers as templates. The HMG I deletions were generated by PCR using the appropriate primers, and their integrity was verified by DNA sequencing. The PCR products were cloned in both pGEX2T (Pharmacia) and pRSETA (Invitrogen) vectors in frame with the glutathione S-transferase (GST) and six-His moieties, respectively. The pPAC expression constructs were generated by cloning the corresponding HMG I derivatives at the BamHI site of the pPAC vector (22). Protein expression and purification. The GST-HMG I deletions were expressed in the HB101 bacterial strain and purified as follows. An exponentially growing 500-ml culture was induced by the addition of isopropyl-b-D-thiogalactopyranoside (IPTG) (0.5 mM final concentration) at an optical density at 600 nm of 0.5 and grown for an additional 2 h at 30°C. The cells were harvested by

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FIG. 3. Identification of functionally distinct DNA binding domains in HMG I. (A) Results of an EMSA experiment using an oligonucleotide spanning the PRDII element of the IFN-b promoter and the indicated HMG I derivatives. The reaction mixtures contained 1 ng of probe and 100 ng of protein either in the absence (2) or in the presence (1) of 100 ng of poly(dG:dC) as a nonspecific competitor. Lanes 21 to 28 originate from a different gel. (B) Results of a quantitative DNase I footprinting experiment using increasing amounts of fulllength HMG I and the deletion derivative lacking the carboxyl acidic tail (1-90 derivative). The full-length HMG I protein binds specifically to the IFN-b promoter (the binding sites are indicated to the left of the gel [thick lines]), while the 1-90 derivative contacts DNA nonspecifically. The amounts of protein used were 2, 5, 15, 30, 60, 125, 250, and 500 ng. (C) Drosophila Schneider cells were cotransfected with an IRE-CAT-based reporter plasmid and 10 ng of an equimolar mixture of pPAC p50- and p65-expressing plasmids (lanes 2, 4, 6, and 8) in the presence (1) or absence of 2 mg of pPAC HMG I derivative-expressing plasmids. Forty-eight hours posttransfection, the cells were harvested and the CAT activity was determined. Shown are the results one of four independent experiments. The variability from experiment to experiment was ,20%. On the right is a Western blot probed with the HMG I(Y) antibody from whole-cell extracts prepared from untransfected Schneider cells (lane 1), cells transfected with wild-type HMG I(1-107) (lane 2), HMG I(1-90) (lane 3), or HMG I(31-107) (lane 4), or 10 mg of HeLa nuclear extract used as a positive control (lane 5). Of note, the 1-90 derivative shows anomalous electrophoretic mobility (migrates faster) due to the deletion of the acidic tail. previously (37, 39) by using gel-purified probes. EMSA results and DNase I footprinting gels were quantitated after scanning with Adobe Photoshop software.

RESULTS

centrifugation and resuspended in 20 ml of ice-cold phosphate-buffered saline supplemented with 10% glycerol, 10 mg of pepstatin per ml, 10 mg of aprotinin per ml, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1% Nonidet P-40. The cells were lysed by sonication, and the insoluble debris was removed by centrifugation at 10,000 rpm for 30 min. The GST-fused proteins were purified by adding 1 ml of glutathione beads to the supernatant. The samples were rotated for 1 h at 4°C, and the beads were precipitated and washed five times with lysis buffer. The glutathione bead-bound proteins were eluted by the addition of free glutathione (4 mM). The six-His-fused HMG I proteins were expressed and purified as previously described (40). DNA-protein interactions. Electrophoretic mobility shift assays (EMSAs) and DNase I and methylation interference experiments were performed as described

Cooperative binding of HMG I(Y) to the IFN-b enhancer. To investigate the interaction between HMG I(Y) and the IFN-b enhancer, we carried out quantitative DNase I footprinting experiments using purified recombinant HMG I and either the wild-type IFN-b enhancer or enhancers bearing specific mutations in each distinct HMG I binding site as probes. HMG I was expressed in bacteria as either a six-His or a GST fusion protein. The addition of increasing amounts of six-His– HMG I to the wild-type IFN-b enhancer results in the protection of five regions within the regulatory element from DNase I cleavage (Fig. 1A). HMG I binds specifically to PRDII (37), to two regions at PRDIV (8, 9), to a portion of the NRDI element located immediately downstream of PRDII (15), and to the TATA box (Fig. 1A). Identical results were obtained when a GST-HMG I fusion protein was used (data not shown). Moreover, all these sites are filled at the same concentration of HMG I, suggesting that the protein binds cooperatively to the five sites or that it binds to these five different sites with equal

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affinity. To distinguish between these two possibilities, we performed additional DNase I footprinting experiments using IFN-b enhancers bearing mutations in the individual HMG I sites as probes. With each mutation, HMG I binding to the corresponding site was abolished, as expected (Fig. 1B to E), but a decrease in HMG I binding to the other sites which are separated from the mutation was also observed. For example, a mutation that converts the PRDII element to the immunoglobulin NF-kB site (IgkB), which does not bind HMG I (37), resulted in a strong decrease of HMG I binding not only to this site but also to NRDI as well as to both sides of PRDIV (Fig. 1D). An identical result was obtained with the mutation at the NRDI element (Fig. 1E). Furthermore, mutation at the HMG I site located at the 59 side of PRDIV not only affected binding to this site but also decreased binding to the 39 side of PRDIV. However, the effect of this mutation on HMG I binding at PRDII, NRDI, and the TATA box was marginal (Fig. 1B). Similar results were obtained with the mutation at the 39 side of PRDIV (Fig. 1C). The biological significance of these observations is demonstrated by the inability of these templates to respond efficiently to virus infection (9, 37; see also below). Since none of these mutations affects binding of any of the known IFN-b gene activators (9, 37), we suggest that highaffinity cooperative binding of HMG I is required for high levels of IFN-b gene activation. Additional evidence for the cooperative nature of HMG I binding was provided by EMSA experiments in which we showed that each of these sites when tested in isolation is bound by HMG I with different affinities (data not shown). For example, 50 times more HMG I protein is required for equivalent binding to the PRDIV site and to PRDII (data not shown). These results, taken together with those of the DNase I footprinting experiments, suggest that HMG I binds to the IFN-b promoter in a highly cooperative manner. HMG I(Y) contains functionally distinct DNA binding domains. To investigate the basis of the HMG I(Y) cooperative DNA binding, we generated a series of deletion mutants of

HMG I (Fig. 2A) which were examined for sequence-specific binding to DNA. These proteins were expressed in Escherichia coli and purified to near homogeneity as either GST or sixhistidine fusions. A representative Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel containing the GSTHMG I deletions is shown in Fig. 2B. These derivatives were tested along with the wild-type protein for sequence-specific binding to the PRDII element. The specificity of DNA binding was examined by the inclusion of the nonspecific competitor poly(dG:dC) in the binding reaction mixture, by DNase I footprinting, and by competitions with specific and nonspecific oligonucleotides. The results of these experiments are shown in Fig. 3A and summarized in Fig. 2A. Consistent with previous results (37), full-length HMG I binds specifically to PRDII, since the addition of the nonspecific competitor did not significantly affect DNA binding (Fig. 3A, lanes 19 and 20 and lanes 21 and 22). Surprisingly, we found that each of the basic repeats differed in its ability to bind DNA specifically. The first repeat binds to DNA with a very low affinity and nonspecifically (Fig. 3A, lanes 11 to 14), the third repeat binds to DNA only nonspecifically (lanes 23 to 26), and only the middle repeat interacts specifically with DNA (lanes 27 and 28). Since the carboxyl-terminal acidic tail is highly conserved among all HMG proteins (5), we tested an HMG I derivative, HMG I(1-90), lacking the 17 carboxyl-terminal acidic amino acids. As shown in Fig. 3A (lanes 3 and 4), this derivative contacts DNA largely nonspecifically, although it does contain the middle repeat, which can bind DNA specifically in isolation (lanes 27 and 28). An excellent illustration of this observation is provided by the DNase I footprinting experiment whose results are shown in Fig. 3B. In contrast to the full-length protein (lanes 2 to 9), the 1-90 derivative binds to DNA with an increased affinity but largely nonspecifically, resulting in a complete coating of the probe at higher protein concentrations (lanes 10 to 18). The nonspecific binding of this derivative is evident when a critical protein concentration is achieved (Fig. 3B, compare lanes 14 and 15), suggesting that protein-protein

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interactions may play a role. As is the case with the full-length protein, derivatives containing the second and the third repeats bind specifically only when the carboxyl-terminal tail is present (Fig. 2A and 3A). However, the acidic tail does not alter the binding properties of the third repeat in isolation, since HMG I derivatives containing the last repeat alone with or without the tail bind DNA only nonspecifically (Fig. 3A, compare lanes 23 and 24 with lanes 25 and 26). Identical results were obtained by using the six-His fusion deletion derivatives of HMG I (data not shown). Thus, in the context of the intact protein, and minimally in the context of the middle and third repeats, the acidic tail is required for sequence-specific DNA binding. We propose that the acidic nature of the tail counteracts the nonspecific DNA binding activity of the third basic repeat only in the presence of the middle repeat. To determine whether sequence-specific DNA binding of HMG I is required in vivo for NF-kB-dependent transcriptional activation, we performed cotransfection experiments with Drosophila Schneider cells, which respond to exogenously added HMG I (39). A reporter construct bearing the interferon gene regulatory element (IRE) of the IFN-b enhancer (37), which includes PRDII and NRDI, cloned immediately upstream of the Drosophila alcohol dehydrogenase promoter was cotransfected with a small amount of NF-kB-expressing plasmids in the absence or presence of expression vectors encoding different HMG I derivatives. Figure 3C shows that transfection of 10 ng of NF-kB-expressing plasmids stimulates transcription eightfold. Cotransfection of full-length HMG I or a derivative lacking the first repeat results in 40-fold stimulation of transcription. By contrast, the 1-90 derivative of HMG I did not facilitate activation by NF-kB, even though it is expressed at the same levels as the full-length protein (Western blot [immunoblot] at the right of Fig. 3C). Since the 1-90 derivative binds to DNA largely nonspecifically, we conclude that sequence-specific DNA binding of HMG I in vivo is required for NF-kB-dependent activation from PRDII. A single HMG I molecule contacts two DNA binding sites simultaneously. We have shown that HMG I contains only one sequence-specific DNA binding domain, the middle repeat, and that there are five HMG I binding sites in the IFN-b promoter. These observations are consistent with a requirement for five HMG I molecules to fill all the sites in the IFN-b promoter. To determine the stoichiometry of HMG I molecules on the IFN-b gene promoter, we carried out EMSAs using the IRE fragment (positions 277 to 237, containing two HMG I binding sites) as a probe and increasing amounts of either the wild-type protein or derivatives lacking specific regions of the protein. As shown in Fig. 4A (lanes 1 to 10), increasing amounts of the full-length HMG I protein form a single nucleoprotein complex with this probe even at high protein concentrations when all of the probe is shifted. This result is intriguing because we have shown that there are two HMG I binding sites present in this probe (Fig. 1), and therefore two different complexes were expected. To define the nucleotides involved in the formation of this complex, we performed methylation interference analysis. As shown in Fig. 4B (lanes 1 and 2), methylation of A residues in both PRDII and NRDI in the minor groove interferes with the formation of this complex. Thus, it appears that a single HMG I molecule contacts both sites on the probe. An identical result was obtained with HMG I derivative 31-107, which lacks only the first repeat (Fig. 4A, lanes 25 to 29,3 and B, lanes 3 and 4). In sharp contrast, the HMG I derivatives lacking the last repeat but containing either the middle repeat alone (54-74) or in combination with the first repeat (1-74) form two distinct complexes with this probe (Fig. 4A, lanes 11 to 24). The faster-

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migrating complex appearing at lower protein concentrations is shown by methylation interference analysis to be due to binding at the PRDII site only (Fig. 4B, lanes 7 to 11). At higher protein concentrations, a second, slower-migrating complex which in addition to PRDII involves specific binding at the NRDI site is observed (Fig. 4B, lanes 5 and 6 and lanes 12 and 13). These experiments strongly suggest that the two complexes formed with these deletion derivatives involve one and two molecules, respectively. The higher-affinity PRDII site is filled first, and then the lower-affinity NRDI site is occupied. In addition, the lack of complete protection at the NRDI site in the slower-migrating complex indicates that protein-protein interactions may also be involved in the formation of these complexes. Finally, the combination of the middle and the last repeats in the presence of the acidic tail contacts the IRE in a manner indistinguishable from that of the wild-type protein, suggesting that the middle and last repeats contact PRDII and NRDI simultaneously. Additional evidence for the stoichiometry of HMG I-DNA complexes was provided by the EMSA experiment whose results are shown in Fig. 5. We hypothesized that if a single full-length HMG I molecule or a derivative containing the middle and the last repeats can occupy two binding sites simultaneously, then addition of a derivative containing the middle repeat alone should not result in the formation of nucleoprotein complexes containing both types of molecules. On the other hand, HMG I derivatives of two different lengths lacking the last repeat should form a heterogeneous complex on the IRE probe, since the two protein molecules would occupy the PRDII and NRDI sites independently. In fact, the full-length HMG I(1-107) protein or the 31-107 derivative fails to form a heterogeneous complex with the derivative containing the middle repeat alone (54-74) (Fig. 5, compare lanes 1 to 5 with 6 to 10 and lanes 20 and 21 with lanes 22 and 23). By contrast, the 1-74 derivative forms a complex with the 54-74 protein (Fig. 5, compare lanes 12 to 15 with lanes 16 to 19). Furthermore, we showed that mixing GST-HMG I and six-His–HMG I fulllength proteins did not generate a new complex on this probe (data not shown). Taken together, the results of these experiments are consistent with the fact that a single molecule of HMG I simultaneously occupies the PRDII and NRDI sequences on the same DNA molecule. Intramolecular cooperativity between the first and second repeats at PRDIV. It has previously been shown that the PRDIV element contains a pair of HMG I binding sites which are filled by HMG I in a highly cooperative manner (9) (Fig. 1). To examine whether the mode of HMG I binding at PRDIV is similar to that at IRE, we tested the same HMG I derivatives in EMSA experiments using the PRDIV element (positions 2104 to 281) as a probe. Surprisingly, we found that an HMG I derivative containing the first and second repeats (derivative 1-74), which fills both HMG I sites on the IRE as a dimer (Fig. 6A, lanes 1 to 5), forms only a single complex on PRDIV, suggesting that a single molecule interacts with both HMG I sites on this probe (Fig. 6A, lanes 6 to 10). In addition, two molecules of an HMG I derivative containing the second and third repeats (derivative 31-107) are required to saturate both binding sites on PRDIV (not shown), whereas a single molecule binds to the two sites on the IRE probe (Fig. 5). As expected, the middle repeat alone forms two complexes on PRDIV (data not shown), as it does on the IRE probe (Fig. 4 and 5). Finally, the intramolecular cooperativity between the first and the second repeats leads to high-affinity DNA binding at PRDIV, since this derivative exhibits a 10-fold-higher DNA binding affinity than the 31-107 derivative, which binds as a dimer to both sites on PRDIV (data not shown).

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FIG. 4. Stoichiometry of HMG I-DNA complexes. (A) Results of an EMSA experiment using the IRE fragment (positions 277 to 237) from the IFN-b promoter as a probe with increasing amounts of the indicated recombinant HMG I derivatives. The sequence of the probe and the two HMG I sites within (lowercase letters) are depicted below the autoradiogram. The amounts of protein used were as follows: 1-107, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 ng; 1-74 and 54-74, 10, 20, 40, 80, 160, 320, and 640 ng; and 31-107, 0.5, 1, 2, 4, and 8 ng. (B) Methylation interference analysis of HMG I-DNA complexes. The full-length HMG I or the derivative lacking the first basic repeat binds to the IRE by contacting both PRDII and NRDI (lanes 1 and 2 and lanes 3 and 4, respectively). Complexes corresponding to a single HMG I molecule contact only the PRDII element (lanes 7 to 9 for the 1-74 derivative and lanes 10 and 11 for the 54-74 derivative). However, complexes corresponding to two HMG I molecules involve additional interactions in the NRDI element as well (lanes 5 and 6 and lanes 12 and 13 for the 1-74 and 54-74 derivatives, respectively). The nucleotide sequence and the base pairs involved in the protein-DNA interactions are depicted on the left side of the gel. DNAs extracted from the free and the bound complexes are shown (F and B, respectively).

The role of the intramolecular cooperativity in high-affinity binding of HMG I to the intact IFN-b promoter was further investigated by DNase I footprinting, as shown in Fig. 6B. In this experiment, we compared the affinity of the HMG I de-

rivative containing the sequence-specific DNA binding domain (middle repeat) with that of the intact HMG I protein. The intact HMG I protein binds to the natural IFN-b enhancer cooperatively with an affinity which is 10-fold higher than that

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FIG. 5. Results of an EMSA experiment using the IRE as a probe and the depicted HMG I derivatives either alone or in combinations. The proteins and amounts used are as follows: lanes 1 to 5, 0.5, 1, 2, 4, and 20 ng of full-length HMG I, respectively; lanes 11 and 24, 200 ng of HMG I(54-74); lanes 6 to 10, a mixture of full-length HMG I and the 54-74 derivative; lanes 12 to 15, 50, 100, 200, and 500 ng of the 1-74 derivative; lanes 16 to 19, a mixture of the 1-74 and 54-74 derivatives; lanes 20 and 21, 50 and 100 ng, respectively, of the 31-107 derivative; and lanes 22 and 23, a mixture of the 31-107 and 54-74 derivatives. The novel complex corresponding to the heterodimer formed between the 1-74 and 54-74 derivatives is indicated (arrow).

of the middle repeat alone, which binds only weakly to PRDIV (Fig. 6B). However, the two proteins display comparable affinities for the PRDII probe, which contains a single HMG I binding site (Fig. 3A). Apparently, protein-protein interactions between HMG I molecules do not suffice for high-affinity binding, because this fragment of HMG I is capable of interacting with itself (our unpublished results). Thus, the intramolecular interactions are required for both high-affinity and cooperative DNA binding to the IFN-b enhancer. In summary, these results revealed a previously unrecognized and unexpected mode of DNA binding. A single HMG I molecule can occupy two binding sites simultaneously by contacting DNA with two of the three basic repeats. Moreover, depending on the DNA sequence of the binding sites, the middle repeat cooperates with either the first or the last repeat. These intramolecular interactions lead to high-affinity and cooperative DNA binding. Intramolecular cooperativity depends on the correct helical phasing of the HMG I binding sites. The results described above, taken together with the architecture of the HMG I binding sites in the IFN-b promoter, raised several intriguing questions. For example, projection of the HMG I binding sites on the double DNA helix revealed that all the sites reside on the same side of the helix. Therefore, we can imagine that there are helical constraints for the intramolecular cooperative binding. To examine this possibility, we constructed IFN-b promoters bearing a half or full helical turn of DNA inserted between the HMG I sites at the PRDII and NRDI elements. This region of the promoter that contains the two HMG I sites either in their wild-type configuration or with the inserted sequence was amplified by PCR and used as a probe in EMSA experiments with different HMG I derivatives. Figure 7A

shows that the full-length HMG I protein binds to the probe containing the half helical insertion fivefold more weakly than to the wild-type probe (compare lanes 1 to 5 with lanes 6 to 10). Moreover, DNase I footprinting experiments revealed that HMG I contacts only PRDII and not NRDI in this template (data not shown). Insertion of an additional half helical turn restored most of the binding (Fig. 7A, lanes 11 to 15). As a control, we showed that insertion of a half helical turn of DNA in the irrelevant position between PRDI and PRDII did not affect HMG I binding (Fig. 7A, lanes 26 to 30). As a further test for the role of intramolecular cooperativity in high-affinity DNA binding of HMG I, we showed that mutations in either PRDII or NRDI dramatically decreased binding (Fig. 7A, lanes 16 to 20 and 21 to 25). The affinity of HMG I for either of the mutated templates parallels its affinity for the PRDIINRDI probe with a 5-bp insertion. Thus, a direct mutation in either binding site and positioning of the binding sites in the opposite side of the DNA helix have similar qualitative effects. Importantly, HMG I binds to the IgkB-mutated probe (PII3 kB) two- to threefold more weakly than to the NRDI mutant (Fig. 7A, compare lanes 16 to 20 with lanes 21 to 25), again confirming that PRDII is a higher-affinity binding site for HMG I than NRDI. Similar results were also obtained when we used HMG I-C or the HMG I derivative lacking the first repeat (data not shown). In contrast, the HMG I derivative containing the middle repeat alone bound with comparable affinities to the wild-type and the helically permutated probes (Fig. 7B). Mutations in either the PRDII or the NRDI element only slightly decreased DNA binding (3- and 1.5-fold, respectively) (Fig. 7B, lanes 16 to 25). From these experiments, we conclude that high-affinity DNA binding of a single wild-type

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FIG. 6. (A) Intramolecular cooperative binding of HMG I at PRDIV requires the first and the second basic repeats. Shown are the results of an EMSA experiment using the IRE (lanes 1 to 5) and PRDIV (lanes 6 to 10) as probes and increasing amounts of the 1-74 HMG I derivative. The amounts of protein used in each case were 10, 25, 50, 100, and 200 ng. (B) Intramolecular cooperativity is required for high-affinity binding of HMG I to the natural IFN-b promoter. Shown are the results of a quantitative DNase I footprinting experiment using the intact IFN-b promoter as a probe and increasing amounts of either the wild-type HMG I protein (1-107) or the derivative containing the middle repeat alone (54-74). The amounts of protein used in each case were 30, 60, 120, 250, 500, 1,000, and 2,000 ng. Of note, equal amounts of these two proteins correspond to a molar ratio of 4:1 for the 54-74 and 1-107 HMG I proteins, respectively.

HMG I molecule to the PRDII-NRDI sites requires that the two sites be positioned at the same phase of the DNA helix. To examine whether the intramolecular cooperative DNA binding of HMG I at PRDII and NRDI correlates with transcriptional activation in vivo, we carried out transient-transfection experiments with human HeLa cells, using wild-type and mutated IFN-b enhancers. Figure 7C shows that the wild-type promoter is induced more than 100-fold upon virus infection. However, conversion of PRDII to the IgkB site (37) or mutation of NRDI significantly decreased virus-dependent activation. Notably, insertion of 5 bp between PRDII and NRDI, which decreased HMG I binding in vitro, also reduced virus induction in vivo. Insertion of an additional half helical turn restored most of the virus-dependent activation of the IFN-b promoter. Thus, high-affinity binding of HMG I in vitro fully correlates with transcriptional activation in vivo. The cooperative DNA binding of HMG I to the intact IFN-b enhancer requires the correct helical phasing of PRDIV and

PRDII elements. We have shown that each pair of HMG I sites present at PRDIV and the IRE is contacted by a single molecule of HMG I. This implies that two molecules of HMG I contact all four binding sites present in the virus-inducible enhancer of the IFN-b gene. To provide direct evidence for this, we carried out EMSA experiments using either the intact enhancer or enhancers bearing mutations in individual HMG I sites. Figure 8A (lanes 1 to 5) shows that increasing amounts of HMG I result in the formation of two distinct complexes with this probe. Mutations at any of the HMG I binding sites in this probe result in a decrease of the lower complex but most significantly in the disappearance of the upper complex (Fig. 8A, lanes 6 to 35). Interestingly, the upper complex appears in a highly cooperative manner, since a twofold increase in the amount of HMG I (from lane 2 to lane 3) suffices for its formation. Figure 8A (lanes 36 to 40) shows that formation of the upper complex involves protein-protein interactions between HMG I molecules bound at PRDIV and the IRE, since

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FIG. 7. Intramolecular cooperative binding of HMG I at the IRE requires a correct helical phasing of the binding sites. (A and B) Results of EMSA experiments using the probes indicated at the bottom and increasing amounts of the wild-type HMG I protein (1, 2, 5, 10, and 25 ng) and the 54-74 derivative (10, 25, 50, 100, and 200 ng), respectively. (C) Human HeLa cells were transfected with the indicated CAT reporter constructs containing either the wild-type (WT) IFN-b promoter (2110 to 120) or the indicated mutations in the same context. The ratio of CAT activity with and without Sendai virus infection (fold virus induction) is shown on the right (results are the averages of six independent experiments). The variability between individual constructs from experiment to experiment was less than 10%.

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FIG. 7—Continued.

the insertion of a half helical turn of DNA between these two elements inhibited formation of the complex. The DNase I footprinting experiment whose results are shown in Fig. 8B demonstrated that HMG I binding at PRDIV was reduced approximately eightfold in this mutant template, verifying the results of the EMSA experiment (Fig. 8A, lanes 36 to 40). Thus, any mutation of the HMG I binding sites or alteration of their relative spacing drastically decreased binding. Taken together, the results of these experiments are consistent with the idea that two molecules of HMG I contact the four binding sites in the IFN-b gene enhancer and make protein-protein contacts, resulting in high-affinity and cooperative DNA binding. DISCUSSION The first step in the assembly of the IFN-b enhanceosome requires binding of HMG I(Y) to the enhancer (39). Most transcription factors accomplish this step because of their ability to target specific DNA sequences in the genome. However, proteins that bind DNA from the minor groove, such as HMG I(Y), have a low ratio of specific to nonspecific DNA binding. HMG I(Y) can bind to almost any AT-rich sequence longer than 4 or 5 bp (35), implying that the human genome contains approximately 107 to 108 putative HMG I binding sites and that the same number of protein molecules would be required to saturate all the sites in vivo. Clearly, it would be highly disadvantageous for the organism if HMG I(Y) was present in every binding site, including regulatory elements of genes not affected by HMG I(Y). These observations raise the interesting question of how HMG I(Y) is specifically recruited to promoters and enhancers. To address this question, we carried out a structural-functional analysis of HMG I(Y) and correlated the in vitro DNA binding properties of the protein with transcriptional activation in vivo. We found that the highaffinity and specific binding of HMG I(Y) to the IFN-b promoter requires multiple regions of the protein involved simultaneously in both protein-protein and protein-DNA interactions. Thus, randomly spaced pairs of short AT-rich sequences in the genome are low-affinity binding sites for the HMG I proteins. By contrast, AT-rich sequences (synthetic or natural) (25) which lie on the same face of the DNA helix constitute high-affinity binding sites for HMG I proteins.

In this paper, we present a detailed structure-function analysis of the regions of HMG I that mediate its specific, highaffinity binding. We have shown that the middle repeat of HMG I(Y) provides the basis for specific DNA binding and that the presence of the first or third repeat results in highaffinity binding of a single HMG I(Y) molecule to the PRDIV or the PRDII/NRDI site, respectively. The distinct binding properties of the basic repeats can be attributed to differences within the core of the motif (GRGRP in the first repeat, which has the lowest affinity for DNA, compared to PRGRP for the middle and third repeats) as well as the flanking amino acids, which make additional contacts with the DNA (13). Within the protein, the spacing between basic repeats may also play a role in binding site recognition. In HMG I, all three repeats are 30 amino acids apart from center to center, while in HMG Y and HMG I-C the distance between the first and middle repeats is only 19 amino acids (Fig. 2A). In a fully extended configuration, 30 amino acids are barely sufficient to span two helical turns of DNA, as is the case with the mutant IFN-b enhancer containing a 10-bp insertion between PRDII and NRDI (Fig. 7A). HMG Y and HMG I-C, with a shorter distance between the first and second repeats than in HMG I, may have distinct targets in vivo. Different distances between basic repeats are found in chironomous cHMG (three repeats) (7) and Drosophila HMG D1 (10 repeats) (3). Another unexpected feature of HMG I(Y) is the role of the carboxyl-terminal acidic tail in sequence-specific DNA binding. An acidic tail of variable length is also present in all members of the HMG family of proteins (5). Despite its strong acidic nature, this region does not function as an activation domain (37). We found that deletion of the tail slightly increased the affinity of HMG I for DNA and, most importantly, impaired its capacity for sequence-specific binding. Other studies have shown that removal of the acidic tail also increases the ability of HMG I to introduce negative supercoils (30). The negative charge of the tail may neutralize nonspecific electrostatic interactions between the third basic repeat and the DNA, since deletion of the tail in the context of the full-length protein or a derivative containing the middle and the last repeats reduces the specific binding of the protein. Interestingly, embedded in this carboxyl-terminal acidic motif is a consensus recognition sequence for casein kinase II, which phosphorylates HMG I in vitro and in vivo (31). IL-4 induces casein kinase II-dependent

FIG. 8. (A) Two molecules of HMG I (arrows) contact all four binding sites in the IFN-b gene enhancer. Results of an EMSA experiment using increasing amounts of recombinant HMG I (0.05, 0.1, 0.2, 0.4, and 1 ng) and the probes indicated at the top are shown. The sequence of each probe and the site of the mutation are shown below the gel. The HMG I sites are indicated (thick lines above the sequences). WT, wild type; 3, sites of mutations. (B) High-affinity and cooperative binding of HMG I to the wild type but not to the helically permutated IFN-b promoter. Shown are results of a quantitative DNase I footprinting experiment using the wild-type IFN-b promoter or a derivative containing a half helical turn of DNA inserted between PRDI and PRDII along with increasing amounts of recombinant HMG I (10, 30, 100, 250, 500, and 1,000 ng).

phosphorylation of HMG I in vivo, decreasing its affinity for DNA (42). Thus, phosphorylation may represent an important means of altering the in vivo function of HMG I. In support of the functional importance of the acidic tail, numerous benign tumors in humans, such as lipomas, involve chromosomal translocations in which the carboxyl-terminal acidic tails of HMG I(Y) and HMG I-C have been deleted (2, 34). The truncated HMG I proteins presumably bind DNA nonspecifically, thus altering gene expression and resulting in neoplastic transformation. A model of HMG I(Y) binding to the IFN-b enhancer is shown in Fig. 9. We have classified the binding sites as two distinct clusters: cluster I includes PRDIV and is contacted by the first and second repeats of one HMG I molecule, while cluster II includes the IRE (the PRDII and NRDI HMG I binding sites) and is contacted by the middle and third repeats of a second HMG I molecule. Within each of the clusters, intramolecular cooperative binding is mediated by a specific pair of basic repeats. However, between the two clusters, intermolecular cooperative binding is mediated by protein-protein interactions between the HMG I(Y) molecules. In the context of the IFN-b enhancer, mutations in the AT-rich sequences in either cluster or the insertion of a half helical turn of DNA between the two clusters impairs both cooperative DNA binding in vitro and virus inducibility in vivo. This pattern of AT-rich sequences on the same face of the DNA helix is repeated in other known in vivo targets of HMG I(Y), such 3660

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3. 4. 5. 6. 7.

FIG. 9. A hypothetical model for HMG I binding at the IRE and PRDIV. A single molecule of HMG I contacts simultaneously the two binding sites present in the IRE (outlined bases). The middle repeat contacts PRDII, whereas the third repeat makes specific contacts at NRDI. However, it is equally possible that the middle repeat contacts NRDI, whereas the third repeat contacts PRDII. The effect of the tail on sequence-specific binding is illustrated (arrow). By contrast, the first and second repeats are involved in simultaneous contacts at PRDIV.

8. 9. 10. 11.

as the E-selectin and class II HLA-DRA gene promoters (1, 43). The distinct arrangement of HMG I(Y) basic repeats on PRDIV and the IRE also indicates that different protein-protein interaction domains can be presented by HMG I(Y) to proximally bound transcription factors, depending on the sequence context. Indeed, we have identified two independent protein-protein interaction surfaces in HMG I (unpublished results). Thus, the surface presented from the first and second repeat binding enhances ATF-2/c-Jun binding but inhibits JunD/FosB binding to PRDIV (9), and the surface presented after the middle and third repeats bound to the IRE enhances NF-kB binding (37). Our work suggests that in conjunction with alterations in DNA structure (10), specific protein-DNA interactions that position one HMG I(Y) molecule to the DNA bound to PRDIV and one at the IRE as well as specific protein-protein interactions between HMG I molecules and between HMG I and NF-kB–ATF-2/c-Jun lead to the formation of functional IFN-b enhanceosomes (39). We have provided the first detailed analysis of the domains in HMG I(Y) required for recognition of biologically important DNA binding sites, which can be extended to studies of the many genes regulated by HMG I(Y) and of proteins containing similar basic repeats. ACKNOWLEDGMENTS This research was initiated in the laboratory of Tom Maniatis, to whom we are grateful for providing important comments and resources. We thank S. C. Chan and D. Petry for their contribution at the early stages of this work and G. Chen for excellent technical assistance. We are grateful to J. Falvo and T. Maniatis for stimulating discussions and comments on the manuscript. We thank T. Abel, A. Aggarwal, M. Gottesman, and D. Hirsh for critical reading of the manuscript. This work was supported by grants awarded to D.T. from the March of Dimes, the Pew Scholars Program in Biomedical Sciences, the Irma T. Hirschl Trust, and the NIH (1 RO1 GM54605-1). J. Yie and S. Liang contributed equally to this work. REFERENCES 1. Abdulkandir, S. A., S. Krishna, D. Thanos, T. Maniatis, J. L. Strominger, and S. J. Ono. 1995. Functional roles of the high mobility group protein HMG I(Y) and Oct-2A in transcriptional regulation of the HLA-DRA gene:

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