Protein HMG-I Research Articles

Cdc2 and Mitogen-activated Protein Kinases Modulate DNA Binding Properties of the Putative Transcriptional RegulatorChironomus High Mobility Group Protein I

Cells of the dipteran insect Chironomus contain a high mobility group protein that is homologous to the mammalian high mobility group proteins I/Y (HMGI/Y). These proteins facilitate the assembly of higher order nucleoprotein complexes. In proliferating cells, >30% of Chironomus HMGI was found to be phosphorylated. The phosphorylation sites were mapped to Ser3, Ser22, and Ser72 and were found to be substrates for the kinases Cdc2 (and mitogen-activated protein (MAP)), MAP, and Ca2+/phospholipid-dependent protein kinase, respectively. In mitotically arrested cells, the extent of phosphorylation at Ser3 increased, whereas phosphorylation at Ser22 remained unchanged. In nondividing cells, phosphorylation at Ser3 and Ser22 was strongly reduced. The DNA binding affinity of Chironomus HMGI was not influenced by single phosphorylation at Ser3 or Ser22. In contrast, phosphorylation at both of these sites resulted in a 10-fold weakening of the binding activity and altered the mode of protein-DNA interaction. Since both human and murine HMGI/Y proteins, similarly to the insect HMGI protein, possess phosphorylation sites for Cdc2 and MAP kinases that intersperse the AT-hook DNA-binding motifs, our results may reflect a general mechanism that regulates the properties and function of this class of putative transcriptional regulators.

Competition between HMG-I(Y), HMG-1 and histone H1 on four-way junction DNA.

High mobility group proteins HMG-I(Y) and HMG-1, as well as histone H1, all share the common property of binding to four-way junction DNA (4H), a synthetic substrate commonly used to study proteins involved in recognizing and resolving Holliday-type junctions formed during in vivo genetic recombination events. The structure of 4H has also been hypothesized to mimic the DNA crossovers occurring at, or near, the entrance and exit sites on the nucleosome. Furthermore, upon binding to either duplex DNA or chromatin, all three of these nuclear proteins share the ability to significantly alter the structure of bound substrates. In order to further elucidate their substrate binding abilities, electrophoretic mobility shift assays were employed to investigate the relative binding capabilities of HMG-I(Y), HMG-1 and H1 to 4H in vitro. Data indicate a definite hierarchy of binding preference by these proteins for 4H, with HMG-I(Y) having the highest affinity (Kd approximately 6.5 nM) when compared with either H1 (Kd approximately 16 nM) or HMG-1 (Kd approximately 80 nM). Competition/titration assays demonstrated that all three proteins bind most tightly to the same site on 4H. Hydroxyl radical footprinting identified the strongest site for binding of HMG-I(Y), and presumably for the other proteins as well, to be at the center of 4H. Together these in vitro results demonstrate that HMG-I(Y) and H1 are co-dominant over HMG-1 for binding to the central crossover region of 4H and suggest that in vivo both of these proteins may exert a dominant effect over HMG-1 in recognizing and binding to altered DNA structures, such as Holliday junctions, that have conformations similar to 4H.

Conformational Changes of DNA Induced by Binding ofChironomus High Mobility Group Protein 1a (cHMG1a): REGIONS FLANKING AN HMG1 BOX DOMAIN DO NOT INFLUENCE THE BEND ANGLE OF THE DNA

High mobility group (HMG) proteins are thought to facilitate assembly of higher order chromatin structure through modulation of DNA conformation. In this work we investigate the bending of a 30-base pair DNA fragment induced by Chironomus HMG1 (cHMG1a), and HMGI (cHMGI) proteins. The DNA bending was measured in solution by monitoring the end-to-end distance between fluorescence probes attached to opposite ends of the DNA fragment. The distance was measured by fluorescence energy transfer using a novel europium chelate as a fluorescence donor. These measurements revealed that the end-to-end distance in the 30-base pair DNA was decreased from approximately 100 A in free DNA to approximately 50.5 A in cHMG1a. DNA complex. The most probable DNA bending angle consistent with these distance measurements is about 150 degrees. The deletion of the charged regulatory domains located close to the C terminus of the HMG1 box domain of cHMG1a protein had no effect on the induced bend angle. The ability to induce a large DNA bend distinguishes the cHMG1 from the cHMGI protein. Only small perturbation of the DNA conformation was observed upon binding of the cHMGI protein. A strong DNA bending activity of cHMG1a and its relative abundance in the cell suggests that this protein plays a very important role in modulation of chromatin structure.

Constitutive activation of Epstein-Barr virus (EBV) nuclear antigen 1 gene transcription by IRF1 and IRF2 during restricted EBV latency.

The Epstein-Barr virus (EBV) EBNA1 gene promoter active in the type I program of restricted viral latency was recently identified and shown to reside in the viral BamHI Q fragment. This promoter, Qp, is active in a wide variety of cell lines and has an architecture reminiscent of eukaryotic housekeeping gene promoters (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995; B. C. Schaefer, J. L. Strominger, and S. H. Speck, Mol. Cell. Biol. 17:364-377, 1997). Here we demonstrate by deletion analysis that the important cis-acting elements regulating Qp are clustered in a relatively small region (ca. 80 bp) surrounding the site of transcription initiation. Immediately upstream of the site of initiation is a region which is protected from DNase I digestion by crude nuclear extracts. Electrophoretic mobility shift analyses (EMSA) employing probes spanning this region demonstrated the presence of two major protein complexes. Deletion analysis of Qp demonstrated that at least one of these complexes plays an important role in Qp activity. Evidence that interferon response factor 2 (IRF2) is a major constituent of the most prominent EMSA complex and that IRF1 may be a minor component of this complex is presented. Transfections into IRF1-/-, IRF2-/-, and IRF1,2-/- fibroblasts demonstrated that absence of both IRF1 and IRF2 reduced Qp activity to approximately the same extent as mutation of the IRF-binding site in Qp, strongly implicating IRF2, and perhaps IRF1, in the regulation of Qp activity. Notably, transcription from Qp was not inducible by either alpha or gamma interferon in EBV-negative B cells but rather was shown to be constitutively activated by IRF1 and IRF2. This observation suggests that IRF1 and IRF2 have a previously unrecognized role as constitutive activators of specific genes. Additionally, data presented indicate that a protein complex containing the nonhistone architectural protein HMG-I(Y) binds to the region identified as the major transcription initiation site for Qp. This observation raises the possibility that HMG-I(Y)-induced DNA bending plays a role in the initiation of transcription from Qp.

Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene.

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor.

An IL-2 response element in the human IL-2 receptor alpha chain promoter is a composite element that binds Stat5, Elf-1, HMG-I(Y) and a GATA family protein.

Expression of the human interleukin-2 (IL-2) receptor alpha chain gene is potently upregulated by its own ligand, IL-2. In this study, we characterize an essential upstream IL-2 response element that contains both consensus and non-consensus GAS motifs, two putative Ets binding sites (EBS), one of which overlaps the consensus GAS motif, and a GATA motif, which overlaps the non-consensus GAS motif. We demonstrate that although the individual components of this element do not respond to IL-2, together they form a composite element capable of conferring IL-2 responsiveness to a heterologous promoter. Multiple factors including Stat5, Elf-1, HMG-I(Y) and GATA family proteins bind to the IL-2 response element and mutation of any one of these binding sites diminishes the activity of this element. An unidentified Ets family protein binds to the EBS overlapping the consensus GAS motif and appears to negatively regulate the human IL-2R alpha promoter. Thus, IL-2-induced IL-2R alpha promoter activity requires a complex upstream element, which appears to contain binding sites for both positive and negative regulatory factors.

Multivalent DNA-binding properties of the HMG-1 proteins.

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements.

Mechanisms of Murine RANTES Chemokine Gene Induction by Newcastle Disease Virus

We have previously defined the lipopolysaccharide (LPS)-responsive element (LRE) in the promoters of murine RANTES (regulated on activation normal T-cell expressed) (MuRantes) and murine IP-10/crg-2, chemokines which have potent chemotactic properties for inflammatory cells including monocytes and T lymphocytes. In the present work, we studied the transcriptional mechanism of MuRantes gene induction by virus and compared it with that of LPS in an effort to understand the host responses to virus and bacterial toxins at the molecular level. MuRantes mRNA expression is induced by Newcastle disease virus (NDV) and LPS in the RAW 264.7 macrophage cell line and peritoneal macrophages of LPS-responsive C3HeB/FeJ mice. In LPS-hyporesponsive C3H/HeJ mice, only NDV induces this chemokine gene, indicating that the pathways of transcriptional activation by NDV and LPS are not identical. Using a transient transfection assay, the minimal virus-responsive element (VRE) was localized between nt -175 and -116. The VRE contains previously defined LRE motif 1 (TCAYRCTT) and motif 3 ((T/A)GRTTTCA(G/C)TTT), which were shown to also be important for initiation of transcription by virus. NDV-stimulated nuclear extracts were tested for trans-activating factors able to bind the VRE. The chromosomal protein HMG-I(C) was shown to bind the 3'-A.T-rich domains of the VRE, and the presence of HMG-I(C) was demonstrated in the VRE-protein complex formed with nuclear extracts from NDV-stimulated, but not unstimulated cells. These findings demonstrate the role of HMG-I(C) in activation of MuRantes promoter by NDV.

Human papillomavirus type 16 E6 protein up-regulates the expression of the high mobility group protein HMG-I(Y) gene in mouse 10T1/2 cells

Using a differential hybridization technique, we have identified a mouse cellular gene, high mobility group protein HMG-I(Y), whose expression is up-regulated by the E6 protein of human papillomavirus (HPV) type 16. This gene was overexpressed in E6-expressing mouse 10T1/2 cells, but not in G418-resistant 10T1/2 cells. The expression of the HMG-I(Y) gene was up-regulated by the transient expression of E6 from a zinc-inducible human metallothionein-IIA gene promoter. Expression was found to be more efficient at a confluent cell density than at a subconfluent cell density. The up-regulation of HMG-I(Y) gene expression by E6, in particular at a confluent cell density, may be part of an altered genetic program in host cells infected with HPV-16.

Structural and functional properties of linker histones and high mobility group proteins in polytene chromosomes.

Variants of histone H1 and high mobility group (HMG) proteins and their genes in Dipteran insects are being studied in our laboratory and have revealed different properties of DNA binding and intrachromosomal distribution. One of the H1 variants of Chironomus is found only in a minority of polytene chromosome bands and differs from the other H1 proteins of the same organism by genomic organization and by an inserted structural motif, the KAPKAP repeat, that is present also in single H1 variants of other, evolutionarily remote organisms. NH2-terminal peptides containing the KAPKAP repeat were found in vitro to interact with DNA, whereas no DNA interaction was observed with the homologous peptide of another H1 variant that does not contain the inserted KAPKAP repeat. We assume that H1 variants containing the KAP motif may interact with a stretch of linker DNA and package chromatin more tightly than other H1 variants. A large series of antibodies directed against different sites in all regions of the H1 molecule is being applied in studying the sites of interaction of the H1 molecule with other molecules in interphase chromatin in terms of antibody epitope accessibility. A search for insect proteins that share properties of the mammalian HMG proteins resulted in isolation and sequencing of two different HMG1 proteins and an HMGI protein. The HMG1 protein of the midge, Chironomus tentans, show a differential distribution in chromosomes. The more abundant cHMG1a protein appears uniformly distributed, whereas the less abundant cHMG1b protein could be localized only in chromosomal puffs. This strongly indicates that these highly similar proteins have different functions in chromatin. The Chironomus HMGI protein and the intron/exon organization of its gene were found to be very similar to human HMGI/Y proteins that are highly abundant in rapidly proliferating cells. Common properties of HMG1 and HMGI proteins include high affinity interaction with AT-rich DNA, irregular DNA structures, and the capacity to bend DNA. These properties suggest that the HMG proteins may have an architectural role in assembling different types of chromatin.

Substrate structure influences binding of the non-histone protein HMG-I(Y) to free nucleosomal DNA.

High mobility group protein HMG-I(Y) selectively binds to stretches of A.T-rich B-form DNA in vitro by recognition of substrate structure rather nucleotide sequence. Recognition of altered DNA structures has also been proposed to explain the preferential binding of this non-histone protein to four-way junction DNA as well as to restricted regions of DNA on random-sequence nucleosome core particles. Here we describe experiments that examine the influence of intrinsic DNA structure, and of structure imposed by folding of DNA around histone cores, on the binding of HMG-I(Y). As substrates for binding, we chose defined-sequence DNA molecules containing A.T-rich segments demonstrated previously to have very different structures in solution. These segments are either intrinsically bent (phase A.T tracts), flexible (oligo[d(A-T)]), or straight and rigid [oligo(dA).oligo(dT)]. DNase-I and hydroxyl radical footprinting techniques were employed to analyze protein binding to these DNAs either free in solution or when they were reconstituted into monomer or dinucleosomes in vitro. Results indicate that the DNA structure exerts a significant influence on HMG-I(Y) binding both when substrates are free in solution and when they are wrapped into nucleosomal structures. For example, when DNA is free in solution, HMG-I(Y) prefers to bind to the narrow minor groove of A.T sequences but sometimes also binds to certain GpC residues having narrowed major grooves that are embedded in such sequences. On the other hand, depending on the structure and/or orientation assumed by particular A.T-rich segments on the surface of reconstituted histone octamers, HMG-I(Y) binding site selection on individual nucleosomes differs considerably. Two observations are of particular importance: (i) HMG-I(Y) can preferentially bind to certain types of A.T-DNA located on the surface of nucleosomes; and (ii) HMG-I(Y) binding can induce localized alterations in the helical periodicity and/or rotational setting of DNA on the surface of some nucleosomes. The abilities of HMG-I(Y) suggests that in vivo the protein may play an important role in recognizing and altering the structure of localized regions of chromatin.

Independent pathways for de-repression of the mouse Ig heavy chain germ-line ε promoter: an IL-4 NAF/NF-IL-4 site as a context-dependent negative element

The activation of germ-line promoters in the Ig heavy chain loci is regulated by cytokines as part of the regulation of B cell commitment to production of new antibody isotypes. Activation of the germ-line promoter of the epsilon heavy chain locus (Gepsilon) and production of IgE are induced by IL-4 and each is virtually undetectable in the absence of IL-4 or the homologous cytokine IL-13. Basal expression of the Gepsilon promoter is repressed by the non-histone chromosomal protein HMG-I(Y), which also contributes to promoter inducibility, and IL-4 stimulates phosphorylation of the C-terminus of HMG-I(Y) through a rapamycin-sensitive pathway. IL-4 treatment of mouse B cells also induces a Gepsilon DNA binding activity with the properties of IL-4 NAF, which is rapidly induced and requires phosphotyrosine for DNA binding activity. This protein binds to a different site from HMG-I(Y), but the IL-4 NAF/NF-IL-4 binding site also is a negative element more active in repression of basal transcription of the Gepsilon promoter. This site acts as a negative element when transferred to the thymidine kinase promoter, but does not confer inducibility. In contrast to HMG-I(Y), IL-4 NAF/NF-IL-4 activation is refractory to rapamycin but sensitive to genistein. These findings indicate that two independent signal transduction pathways diverge from the IL-4 receptor and suggest that normal expression of Gepsilon RNA or IgE is low in part because the germ-line promoter is kept in a state of repression which requires de-repression through several cooperative pathways. These pathways target conserved nucleotide sequence motifs whose precise function depends on the promoter context in which they are situated.

HMG-I Binds to GATA Motifs: Implications for an HPFH Syndrome

We have examined binding of the nuclear protein HMG-I to the human γ-globin promoter. We find that HMG-I binds preferentially to the more 3′ of a pair of GATA motifs in the γ-globin promoter; this paired motif is bound by the erythroid factor GATA-1. A naturally occurring mutation (−175 T-C) in the area bound by HMG-I results in overexpression of γ-globin in adult red blood cells (HPFH) and up-regulation of the γ-globin promoter in in vitro expression assays; HMG-I does not bind to this mutant sequence. A survey of GATA motifs from other globin cis-elements demonstrates HMG-I binding to most of them. These findings implicate HMG-I in the HPFH phenotype; we speculate that it may participate in the formation of multiprotein complexes that regulate globin gene expression.

Interleukin 4-inducible Phosphorylation of HMG-I(Y) Is Inhibited by Rapamycin

The non-histone chromosomal protein HMG-I(Y) participates in repression of transcription directed by a promoter which confers interleukin 4 (IL-4)-inducible activation in transfected B cell lines. Metabolic labeling, phosphoamino acid analyses, and in vitro phosphorylation studies demonstrate that IL-4 induces serine phosphorylation of HMG-I(Y) in B lymphocytes. Phosphopeptide mapping shows that the predominant site of phosphorylation contains a casein kinase II consensus motif. The immunosuppressive agent rapamycin has been shown preferentially to inhibit IgE production by IL-4-treated human B cells. It is shown here that rapamycin inhibits both activation of the human germ line epsilon promoter by IL-4 and IL-4-inducible phosphorylation of HMG-I(Y). These findings demonstrate a rapamycin-sensitive pathway that transduces signals from the IL-4 receptor to nuclear factors that regulate inducible transcription. The affinity of normal nuclear HMG-I(Y) for DNA is increased by dephosphorylation in vitro, whereas in vitro kinase reactions using casein kinase II decrease recombinant HMG-I(Y) binding to DNA. These data further suggest a novel mechanism in which phosphorylation triggered by IL-4 or other cytokines could regulate the effects of HMG-I(Y) on gene transcription.

Mutation responsible for the mouse pygmy phenotype in the developmentally regulated factor HMGI-C.

Growth is one of the fundamental aspects in the development of an organism. Classical genetic studies have isolated four viable, spontaneous mouse mutants disrupted in growth, leading to dwarfism. Pygmy is unique among these mutants because its phenotype cannot be explained by aberrations in the growth hormone-insulin-like growth factor endocrine pathway. Here we show that the pygmy phenotype arises from the inactivation of Hmgi-c (ref. 6), a member of the Hmgi family which function as architectural factors in the nuclear scaffold and are critical in the assembly of stereospecific transcriptional complexes. Hmgi-c and another Hmgi family member, Hmgi(gamma) (ref. 10), were found to be expressed predominantly during embryogenesis. The HMGI proteins are known to be regulated by cell cycle-dependent phosphorylation which alters their DNA binding affinity. These results demonstrate the important role of HMGI proteins in mammalian growth and development.

Differential Regulation of a Multipromoter Gene.: SELECTIVE 12-O-TETRADECANOYLPHORBOL-13-ACETATE INDUCTION OF A SINGLE TRANSCRIPTION START SITE IN THE HMG-I/Y GENE

The human HMG-I/Y gene, encoding the non-histone "high mobility group" proteins HMG-I and HMG-Y, is transcriptionally activated in human K562 erythroleukemia cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induces differentiation of K562 cells within 2-4 days after treatment. In this report, we show that transcriptional activation of the HMG-I/Y gene is dependent on protein synthesis and is an early event (2 h after induction) in the TPA-mediated differentiation process. Of the four functional transcription start sites present in the gene, only one (start site 2) is preferentially induced upon TPA treatment. This is the first report, to our knowledge, of the preferential utilization of a specific transcription start site in response to a particular stimulus in a gene that contains multiple promoters. This indicates that each start site in the gene has the potential to be independently regulated instead of being coordinately controlled as shown in a number of other genes. In addition, sequences upstream of the inducible start site, which contains a TPA-responsive element, mediates TPA inducibility through AP1 (or an AP1-like) transcription factor. The HMG-I/Y proteins function as key regulators of gene expression and play a significant role in chromatin structural changes as well. The cloning and sequence analyses previously reported indicated the structure of the HMG-I/Y gene to be highly complex and predicted its expression to be tightly regulated. The results presented here confirm and extend these earlier findings.

The non‐histone chromosomal protein HMG‐I(Y) contributes to repression of the immunoglobulin heavy chain germ‐line ε RNA promoter

The rate of germ-line RNA transcription correlates with the rate of immunoglobulin heavy chain isotype switching. A promoter element for the transcription of RNA from the germ-line mouse immunoglobulin epsilon heavy chain constant region gene is induced by interleukin(IL)-4 and lipopolysaccharide, and is bound at its transcription initiation sites by an IL-4-inducible nuclear protein, NF-BRE. To examine the function of the binding site for this IL-4-inducible complex, substitution mutations were introduced in the promoter. These binding site mutations increased promoter activity and decreased binding of NF-BRE. To investigate the paradox of an IL-4-inducible protein binding to a repressor site in an IL-4-inducible promoter, we determined that the non-histone chromosomal protein HMG-I(Y) binds at the transcription initiation sites of the germ-line epsilon promoter. Assays with antisera against HMG-I(Y) revealed monomeric HMG-I(Y) in nuclear extracts. Cotransfection of an expression construct directing the synthesis of anti-sense HMG-I(Y) RNA also increased promoter activity, consistent with a repressor function of HMG-I(Y). Thus, the data are most consistent with a model in which HMG-I(Y) participates in repression of promoter activity. The effects of IL-4 may include derepression at this site.

Replacement of Conserved Threonines by Alanine Residues in High Mobility Group Protein HMG-I(Y): Effect on DNA Binding Affinity

A threonine residue at the beginning of each DNA-binding domain of HMG-I (residue numbers 21, 53, and 78) is conserved among mammalian species and proposed to help stabilize the A·T-hook DNA-binding motif. Phosphorylation of threonines number 53 and 78 of human HMG-I(Y) both in vivo and in vitro leads to a 20 fold reduction in the proteins DNA binding affinity. Recombinant human HMG-I proteins were engineered to contain alanine instead of the conserved threonine in each DNA-binding domain. The DNA dissociation constant of each protein was assayed at various salt concentrations by competition with the fluorescent dye Hoechst 33258 for an AT-rich DNA substrate. Replacement of these threonines did not affect the equilibrium binding of these proteins to DNA as compared with wild-type HMG-I and HMG-Y. Molecular modelling of analogous peptides supported this finding. We conclude that these threonines are not directly important for A·T-hook DNA-binding and are conserved phosphorylation sites for down regulation of DNA binding by the A·T-hook motif in the HMG-I(Y) proteins.

Cell cycle regulation and functions of HMG-I(Y).

Members of the HMG-I(Y) family of "high mobility group" (HMG) proteins are distinguished from other nonhistone chromatin proteins by their ability to preferentially recognize the structure of the narrow minor groove of A.T-sequences of B-form DNA. In vivo the HMG-I(Y) proteins are localized in the A.T-rich G/Q bands and in the "scaffold-associated regions" (SARs) of metaphase chromosomes. These proteins also share with some of the other "HMG box" proteins the ability to recognize non-B-form structures, such as cruciforms (four-way junctions), as well as the possessing the capacity to introduce both bends and supercoils in substrate DNAs. These characteristics, along with their ability to specifically interact with a number of known transcription factors, enable the HMG-I(Y) proteins to function in vivo as structural transcription factors for a number mammalian genes. The HMG-I(Y) proteins are also in vivo substrates for the cell cycle regulated Cdc2 kinase which phosphorylates the DNA-binding domain(s) of the protein and, as a result, decreases their substrate binding affinity. This reversible in vivo pattern of Cdc2 kinase phosphorylations during the cell cycle is likely to play a major role in mediating the biological function(s) of the HMG-I(Y) proteins.

HMGI(Y) and Sp1 in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene.

Expression of the chemokine MGSA/GRO is upregulated as melanocytes progress to melanoma cells. We demonstrate that constitutive and cytokine induced MGSA/GRO alpha expression requires multiple DNA regulatory regions between positions -143 to -62. We have previously shown that the NF-kappa B element at -83 to -65 is essential for basal and cytokine induced MGSA/GRO alpha promoter activity in the Hs294T melanoma and normal retinal pigment epithelial (RPE) cells, respectively. Here, we have determined that the Sp1 binding element located approximately 42 base pairs upstream from the NF-kappa B element binds Sp1 and Sp3 constitutively and this element is necessary for basal MGSA/GRO alpha promoter activity. We demonstrate that the high mobility group proteins HMGI(Y) recognize the AT-rich motif nested within the NF-kappa B element in the MGSA/GRO alpha promoter. Loss of either NF-kappa B or HMGI(Y) complex binding by selected point mutations in the NF-kappa B element results in decreased basal and cytokine induced MGSA/GRO alpha promoter activity. Thus, these results indicate that transcriptional regulation of the chemokine MGSA/GRO alpha requires at least three transcription factors: Sp1, NF-kappa B and HMGI(Y).