High Mobility Group Protein HMG Research Articles

Specific interaction between H1 histone and high mobility protein HMG1.

High mobility group proteins HMG1 and -2 and histone H1 are structural components of chromatin. Previously, we reported that HMG1 interacts with H1 histone in a way that modulates the ability of H1 to condense DNA in vitro, suggesting that these proteins may act together in vivo to regulate locally the condensation state of chromatin, possibly affecting replication and/or transcription. Here we show that reduced (native) HMG1 binds to H1 cooperatively at pH 6.0 as a tetramer with a dissociation constant of 3.4 x 10(-8) M, and at pH 7.5 as a monomer with a dissociation constant less than 10(-9) M. Denaturation through oxidation of sulfhydryl groups has a strong effect on the interaction of HMG1 with H1 histone, suggesting that the reduced state of HMG1 is critical to its function. Oxidized HMG1 failed to bind H1 at pH 7.5, and its binding at pH 6 was biphasic; the first three (or two) molecules of H1 were bound with a dissociation constant of 2 x 10(-8) M with negative cooperativity, and the last one (or two) H1's were bound cooperatively with KD = 1.8 x 10(-7) M. Regulation of the pH or the concentration of some other ion may be used in vivo to alter the interactions between HMG1 and -2, H1 histone, and DNA.

Mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements

The high mobility group protein HMG I(Y) and the transcription factor NF-κB are required for the activity of positive regulatory domain II (PRDII), a virus-inducible regulatory element of the human interferon-β gene promoter. In this paper we provide evidence that HMG I(Y) is also required for the activity of PRDIV, a regulatory element that synergizes with PRDII. In this case, HMG I(Y) stimulates binding of activating transcription factor 2 (ATF-2) and the assembly of inducible complexes containing ATF-2 and c-Jun. Remarkably, HMG I(Y) also specifically interacts with the leucine zipper/basic region of ATF-2, and ATF-2 in turn interacts with NF-κB. We therefore propose that the HMG I(Y) plays a critical structural role in establishing transcriptional synergy between PRDII and PRDIV by promoting the activities and/or binding of NF-κB and ATF-2 and by facilitating their interaction.

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known. In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion. Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments. Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp. By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations. In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn of DNA compared with substrates active in the presence of HU protein. Analysis of different domains of HMG1 generated by partial proteolytic digestion indicate that DNA-binding domain B is sufficient for both bending and invertasome assembly. We suggest that an important biological function of HMG1 and HMG2 is to facilitate cooperative interactions between cis-acting proteins by promoting DNA flexibility. A general role for HMG1 and HMG2 in chromatin structure is also suggested by their ability to wrap DNA duplexes into highly compact forms.

Multiple closely-linked NFAT/octamer and HMG I(Y) binding sites are part of the interleukin-4 promoter.

We show here that the immediate upstream region (from position -12 to -270) of the murine interleukin 4 (Il-4) gene harbors a strong cell-type specific transcriptional enhancer. In T lymphoma cells, the activity of the Il-4 promoter/enhancer is stimulated by phorbol esters, Ca++ ionophores and agonists of protein kinase A and inhibited by low doses of the immunosuppressant cyclosporin A. The Il-4 promoter/enhancer is transcriptionally inactive in B lymphoma cells and HeLa cells. DNase I footprint protection experiments revealed six sites of the Il-4 promoter/enhancer to be bound by nuclear proteins from lymphoid and myeloid cells. Among them are four purine boxes which have been described to be important sequence motifs of the Il-2 promoter. They contain the motif GGAAA and are recognized by the inducible and cyclosporin A-sensitive transcription factor NFAT-1. Three of the Il-4 NFAT-1 sites are closely linked to weak binding sites of Octamer factors. Several purine boxes and an AT-rich protein-binding site of the Il-4 promoter are also recognized by the high mobility group protein HMG I(Y). Whereas the binding of NFAT-1 and Octamer factors enhance the activity of the Il-4 promoter, the binding of HMG I(Y) suppresses its activity and, therefore, appears to be involved in the suppression of Il-4 transcription in resting T lymphocytes.

The High Mobility Group protein HMG I(Y) is required for NF-κB-dependent virus induction of the human IFN-β gene

In this paper, we show that both NF-κB and the high mobility group protein I(Y) (HMG I(Y)) are required for virus induction of the human interferon-β (IFN-β) gene. NF-κB binds to the terminal regions of a 10 by regulatory sequence through contacts in the major groove, while HMG I(Y) recognizes the central region of the same sequence through contacts in the minor groove. Mutations that interfere with binding of either protein decrease the level of virus induction, and activation of the gene can be blocked by either NF-κB or HMG I(Y) antisense RNA. HMG I(Y) stimulates the binding of NF-κB to the IFN-β promoter, and it may also function as a promoter-specific accessory factor for NF-κB transcriptional activity.

Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2.

The biochemical processes responsible for the recognition and repair of cisplatin-damaged DNA in human cells are not well understood. We have developed a damaged DNA affinity precipitation technique that allows the direct visualization and characterization of cellular proteins that bind to cisplatin-damaged DNA. The method separates damaged DNA-binding proteins from complex radiolabeled cell mixtures and further resolves them into individual polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique is complementary to gel retardation and Southwestern blotting analyses that have been previously used to identify cellular components that specifically bind to cisplatin-damaged DNA. Using this technique, we have characterized a set of HeLaS3 nuclear proteins of 26.5, 28, 90, and 97 kDa that specifically bind to cisplatin-DNA adducts. Competition studies with soluble cisplatin-damaged DNA confirmed these findings. The major cisplatin-damaged DNA-binding proteins of 26.5 and 28 kDa recognized adducts of DNA modified with cisplatin but not with its trans-isomer or with UV radiation. These proteins were purified 450-fold to near homogeneity by ion-exchange and cisplatin-damaged DNA affinity chromatography. Amino-terminal sequence analysis showed that the 26.5- and 28-kDa proteins were identical to high mobility group (HMG) proteins HMG-2 and HMG-1, respectively.

Specific Binding of Chromosomal Protein HMG1 to DNA Damaged by the Anticancer Drug Cisplatin

The mechanism of action of the anticancer compound cis-diamminedichloroplatinum(II) (cisplatin) involves covalent binding to DNA. In an effort to understand the tumor-specific cytotoxicity of such DNA damage, the interactions of these lesions with cellular proteins have been studied. One such protein has been identified as the high-mobility group protein HMG1. Recombinant rat HMG1 binds specifically (dissociation constant 3.7 +/- 2.0 x 10(-7) molar) to DNA containing cisplatin d(GpG) or d(ApG) intrastrand cross-links, which unwind and bend DNA in a specific manner, but not to DNA modified by therapeutically inactive platinum analogs. These results suggest how HMG1 might bind to altered DNA structures and may be helpful in designing new antitumor drugs.

DNA binding properties of an HMG1-related protein from yeast mitochondria.

The DNA binding properties of ABF2, an abundant protein found in the mitochondria of the yeast Saccharomyces cerevisiae have been examined in detail. ABF2 is closely related to the vertebrate high mobility group protein HMG1 and like HMG1, ABF2 will introduce negative supercoils into a relaxed, double-stranded circular DNA molecule in cooperation with a DNA topoisomerase. Additionally, ABF2 binds approximately 5-10 times more tightly to negatively supercoiled DNA than to relaxed circular or linear DNA. Although ABF2 binds to most random double-stranded sequences with roughly equal affinity, its binding within certain key regulatory regions is qualitatively quite different. First, ABF2 binding induces a distinct pattern of DNA bending within the chromosomal origin of DNA replication, ARS1. Second, ABF2 binding to all nuclear replication origins tested, in addition to a critical mitochondrial promoter and replication origin, is clearly nonrandom as visualized by DNase1 footprinting. Analysis of the sequences found within these regions as well as competition experiments with synthetic DNA molecules suggest that site-specific DNA binding may be accomplished by the phased distribution of short stretches of poly(dA), which exclude ABF2 binding. These patterns of ABF2 DNA binding suggest a role for the protein in genome organization and site-specific regulation of transcription or DNA replication.

A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria.

ABF2 (ARS-binding factor 2), a small, basic DNA-binding protein that binds specifically to the autonomously replicating sequence ARS1, is located primarily in the mitochondria of the yeast Saccharomyces cerevisiae. The abundance of ABF2 and the phenotype of abf2- null mutants argue that this protein plays a key role in the structure, maintenance, and expression of the yeast mitochondrial genome. The predicted amino acid sequence of ABF2 is closely related to the high-mobility group proteins HMG1 and HMG2 from vertebrate cell nuclei and to several other DNA-binding proteins. Additionally, ABF2 and the other HMG-related proteins are related to a globular domain from the heat shock protein hsp70 family. ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression.

Cyclic Adenosine 3′, 5′-Monophosphate-Dependent Phosphorylation of HMG 14 Inhibits Its Interactions with Nucleosomes

The high mobility group protein HMG 14, which is preferentially associated with nucleosomes containing active gene sequences, is phosphorylated on different sites according to the tissue and stimulus being studied. In the thyroid, HMG 14 displays TSH-dependent phosphorylation that is mediated by cAMP-dependent protein kinase (A-kinase). We have, therefore, studied how phosphorylation of HMG 14 on its major and minor A-kinase sites (Ser-6 and -24) affects its interactions with nucleosomes and various forms of DNA, since this could reflect a means of regulating its function of binding to active chromatin. Approximately twice as much Ser-6 phospho- and 4 times as much Ser-6,24 diphospho-HMG 14 were required to produce the same degree of nucleosome band displacement as that caused by native unphosphorylated HMG 14. Phosphorylation also reduced the ability of HMG 14 to protect the ends of nucleosomal DNA from thermal denaturation. When the electrophoretic mobility of naked DNA was examined, the Ser-6 phospho-HMG 14 was about half as effective as native HMG 14 in retarding the various forms of double stranded DNA, and Ser-6,24 diphospho-HMG 14 was even less effective. Our data demonstrate that electrostatic interactions between DNA and basic amino acids in two highly conserved regions (residues 1-5 and 16-27) can be modulated by phosphorylation at Ser-6 and Ser-24. The ability of mammalian HMG 14, but not HMG 17, to display hormone-dependent phosphorylation may indicate a route for differentially modulating their binding to transcriptionally active chromatin.

Nucleosome assembly in vitro: separate histone transfer and synergistic interaction of native histone complexes purified from nuclei of Xenopus laevis oocytes.

High speed supernatants of Xenopus laevis oocyte nuclei efficiently assemble DNA into nucleosomes in vitro under physiological salt conditions. The assembly activity cofractionates with two histone complexes composed of the acidic protein N1/N2 in complex with histones H3 and H4, and nucleoplasmin in complex with histones H2B and H2A. Both histone complexes have been purified and their nucleosome assembly activities have been analysed separately and in combination. While the histones from the N1/N2 complexes are efficiently transferred to DNA and induce supercoils into relaxed circular plasmid DNA, the nucleoplasmin complexes show no supercoil induction, but can also transfer their histones to DNA. In combination, the complexes act synergistically in supercoil induction thereby increasing the velocity and the number of supercoils induced. Electron microscopic analysis of the reaction products shows fully packaged nucleoprotein structures with the typical nucleosomal appearance resulting in a compaction ratio of 2.8 under low ionic strength conditions. The high mobility group protein HMG-1, which is also present in the soluble nuclear homogenate from X. laevis oocytes, is not required for nucleosome core assembly. Fractionation experiments show that the synergistic effect in the supercoiling reaction can be exerted by histones H3 and H4 bound to DNA and the nucleoplasmin complexes alone. This indicates that it is not the synchronous action of both complexes which is required for nucleosome assembly, but that their cooperative action can be resolved into two steps: deposition of H3 and H4 from the N1/N2 complexes onto the DNA and completion of nucleosome core formation by addition of H2B and H2A from the nucleoplasmin complexes.

Characterization of cDNA sequences corresponding to three distinct HMG-1 mRNA species in line CHO Chinese hamster cells and cell cycle expression of the HMG-1 gene.

We have isolated cDNA clones encoding the high mobility group (HMG) protein HMG-1 in line CHO Chinese hamster cells. The cDNA clones correspond to the three HMG-1 mRNA species detected on Northern blots. Three different polyadenylation sites are found to be used. The three mRNA species of sizes 1.05, 1.45 and 2.45 kb are generated by differential polyadenylation at sites 115 nucleotides, 513 nucleotides and 1515 nucleotides downstream from the stop codon. A perfectly conserved putative poly(A) signal AAUAAA is present upstream of only one of the three poly(A) sites. Two homologous but imperfect sequences exist upstream from the other two poly(A) sites. All three HMG-1 mRNA species maintain significant levels throughout the M, G1 and S phases of the cell cycle and the rate of large HMG protein (HMG-1 and HMG-2) synthesis increases approximately two-fold from G1 to S phase.

Supercoil-dependent recognition of specific DNA sites by chromosomal protein HMG 2

The ability of the chromosomal high mobility group protein HMG 2 to recognize supercoil-dependent structures within the chicken adult beta-globin gene was investigated by examining its ability to protect such sites from digestion by S1 nuclease. Low molar ratios of HMG 2 were found to be sufficient for complete inhibition of S1 cleavage of a supercoiled plasmid containing the globin gene. Furthermore, HMG 2 protected an S1 cleavage site within the 5'-flanking region of the globin gene, in preference to a palindromic S1 site within the plasmid vector.

Phosphorylation alters the affinity of high mobility group protein HMG 14 for single-stranded DNA

The effect of phosphorylation on the affinity of HMG 14 from calf thymus for single-stranded DNA (ssDNA) was studied, using a cyclic GMP-dependent protein kinase from bovine lung and a nuclear protein kinase II from rat liver. When phosphorylated by G-kinase, HMG 14 eluted at 0.27 M NaCl from the ssDNA-column, whereas the native protein eluted at 0.30 M salt concentration. In contrast, phosphorylation by nuclear protein kinase II did not alter dissociation of HMG 14 from ssDNA and the phosphoprotein consequently coeluted with the native HMG 14. Thus, addition of a negative charge by phosphorylation of the Ser-6 residue by G-kinase presumably weakens the interaction between the DNA-binding amino acids of HMG 14 and the negatively charged phosphate groups of DNA.

RAD6 gene of Saccharomyces cerevisiae encodes a protein containing a tract of 13 consecutive aspartates.

The RAD6 gene of Saccharomyces cerevisiae is required for postreplication repair of UV-damaged DNA, for induced mutagenesis, and for sporulation. We have mapped the transcripts and determined the nucleotide sequence of the cloned RAD6 gene. The RAD6 gene encodes two transcripts of 0.98 and 0.86 kilobases which differ only in their 3' termini. The transcribed region contains an open reading frame of 516 nucleotides. The rad6-1 and rad6-3 mutant alleles, which we have cloned and sequenced, introduce amber and ochre nonsense mutations, respectively, into the open reading frame, proving that it encodes the RAD6 protein. The RAD6 protein predicted by the nucleotide sequence is 172 amino acids long, has a molecular weight of 19,704, and contains 23.3% acidic and 11.6% basic residues. Its most striking feature is the highly acidic carboxyl terminus: 20 of the 23 terminal amino acids are acidic, including 13 consecutive aspartates. RAD6 protein thus resembles high mobility group proteins HMG-1 and HMG-2, which each contain a carboxyl-proximal tract of acidic amino acids.

Interaction between domains in chromosomal protein HMG-1.

Peptides corresponding to the N-terminal, central and central plus C-terminal domains of high mobility group protein HMG-1 from calf thymus have been isolated after digestion in solution with protease V8 under structuring conditions (0.35 M NaCl, pH 7.1). The effect of the interaction of these peptides with DNA on the topological properties of the nucleic acid has been studied and compared with the change in superhelicity produced by the whole protein. It appears that the region responsible for this effect is the central domain of HMG-1. The isolated N-terminal and central domains of this protein maintain their secondary and tertiary structure as observed by spectroscopic techniques. However, when the central domain is covalently linked only to the acidic C-terminal part of the molecule, its secondary and tertiary structures are lost as well as its property to alter DNA superhelicity. The results are discussed in relation to the interactions occurring between the different domains and the possible functional interactions of this protein.

High mobility group proteins HMG1 and HMG2 do not decrease the melting temperature of DNA

High mobility group proteins 1 and 2 isolated in non-denaturing conditions cannot decrease the temperature of denaturation of DNA. When they are isolated or treated with tricloroacetic acid a hyperchromic peak below the melting temperature of free DNA appears in agreement with previous data ( Javaherian et al. (1979) Nucl . Acids Res. 6, 3569-3580). We show that this is due to light scattering of aggregated protein at submelting temperatures and not to melting of DNA.

Release of ribonucleoprotein during digestion of rat testis chromatin with deoxyribonuclease II (3.1.4.6)

1. 1. The composition of rat testis chromatin proteins in fractions produced by limited DNase II digestion followed by differential precipitation with MgCl 2 has been studied. 2. 2. Over 50% of the acid-soluble proteins in the soluble chromatin fraction appeared to be quite similar to proteins which are associated with ribonucleoprotein (RNP) particles in HeLa cells. 3. 3. Although the ratios of the testis RNP protein components differed from those of HeLa RNP particles, the three major polypeptides were most similar to the HeLa components designated A2, B2, and C1. 4. 4.|The soluble chromatin fraction was also enriched in the high mobility group proteins HMG1 and HMG2.

Inhibition of transcription in somatic cells by microinjection of antibodies to chromosomal proteins.

The in vivo function of defined chromosomal proteins was examined by microinjecting purified antibody and antibody fragments into living fibroblasts. The involvement of histones and chromosomal high mobility group proteins HMG-1, 2, and 17 in transcription was visualized by studying the [3H]uridine incorporation in KD human fibroblasts after microinjection of fluoresceinated antibodies to these proteins. Nuclear uridine incorporation was not affected by microinjection of control antibodies or by the presence of immune complexes formed after microinjection of antibodies to chromosomal proteins that are not involved in transcription. In contrast, injection of anti-histone IgG, F(ab')2, or Fab and anti-HMG-17 IgG causes a significant reduction in transcription. The reduction is proportional to the amount of antibody introduced into the cell. We conclude that histones and protein HMG-17 are present on transcribed regions of the genome and that passage of RNA polymerase along the chromatin fiber is prevented by antibody binding to these proteins.

Quantitative changes of high mobility group non-histone chromosomal proteins HMG1 and HMG2 during rooster spermatogenesis

The quantitative changes of a group of non-histone chromosomal proteins identified by its solubility, electrophoretic mobility and amino acid analysis as the high mobility group proteins HMG1 and HMG2, were studied throughout rooster spermatogenesis. The ratio HMG1/HMG2 remained constant (0.66 ± 0.04) during the transition from dividing meiotic and premeiotic cells to nondividing spermatids and from transcriptionally active cells (spermatogonia, spermatocytes and early spermatids) to transcriptionally inactive late spermatids. The ratio HMG1/nucleosomal histone and HMG2/nucleosomal histone increase markedly at the end of spermiogenesis during the transition from nucleohistone to nucleoprotamine when nucleosomes are being disassembled. The high mobility group chromosomal proteins HMG1 and HMG2 were not detectable in the nuclei of rooster spermatozoa.