High mobility group protein D


Covalent modification of HMG proteins

The high mobility group (HMG) 1 and 2 proteins are the most abundant non-histone components of chromosomes. HMG1 proteins are abundant components of chromatin. A subfamily of the HMG proteins containing the HMG1 box domain (HMG1-BD) is widely distributed in eukaryotic cells from yeast to man. The members of this group are thought to have various functions related to modulation of transcription, DNA integration, and recombination. Since these proteins have an ability to induce strong bends and unwind DNA, they are called architectural components of chromatin. The most abundant of the HMG1 box proteins are the HMG1 and HMG2 proteins. They are composed of one or two HMG1-BDs, which are primarily responsible for contacts with DNA. HMG1-BDs are amino- and/or carboxyl-terminally flanked by stretches of positively or negatively charged residues. These regions modulate the binding affinity of HMG1-BDs, but do not influence the extent of DNA distortion. Deletion of these regions, in particular those of the negatively charged carboxyl-terminal tails, alters binding specificity of the HMG1 proteins. Moreover, the C-terminal portion of the HMG1 proteins is important for stimulation of transcription and nuclear retention (Wisniewski, 1999 and references).

Essentially the entire pool of HMG1 proteins in Drosophila embryos and Chironomus cultured cells is phosphorylated at multiple serine residues located within acidic tails of these proteins. The phosphorylation sites match the consensus phosphorylation site of casein kinase II. Electrospray ionization mass spectroscopic analyses reveal that Drosophila HMGD and Chironomus HMG1a and HMG1b are double-phosphorylated and that Drosophila HMGZ is triple-phosphorylated. The importance of this post-translational modification was studied by comparing some properties of the native and in vitro dephosphorylated proteins. It was found that dephosphorylation affects the conformation of the proteins and decreases their conformational and metabolic stability. Moreover, dephosphorylation weakens binding of the proteins to four-way junction DNA by 2 orders of magnitude, whereas the strength of binding to linear DNA remains unchanged. Based on these observations, it has been proposed that the detected phosphorylation is important for the proper function and turnover rates of these proteins. Since the occurrence of acidic tails containing canonical casein kinase II phosphorylation sites is common to diverse HMG and other chromosomal proteins, these results are probably of general significance (Wisniewski, 1999).

The transcriptional coactivators CBP and P/CAF are required for activation of transcription from the IFN beta enhanceosome. CBP and P/CAF acetylate HMG I(Y), the essential architectural component required for enhanceosome assembly. Acetylation takes place at distinct lysine residues, causing distinct effects on transcription. Thus, in the context of the enhanceosome, acetylation of HMG I by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly. Acetylation of HMG I(Y) by CBP is essential for turning off IFN beta gene expression. The acetyltransferase activities of CBP and P/CAF modulate both the strength of the transcriptional response and the kinetics of virus-dependent activation of the IFN beta gene (Munshi, 1998).

Protein interactions of HMG proteins

In vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their varying abilities to inhibit NF-kappaB (Drosophila homolog: Dorsal) binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. The inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes (Tran, 1997).

The DNA-dependent protein kinase (DNA-PK) holoenzyme consists of a 470-kDa catalytic subunit (DNA-PKcs), a DNA-binding regulatory component known as Ku protein, and double-stranded DNA (dsDNA) with ends. The activity of DNA-PK in vitro is stimulated by non-histone chromosomal high mobility group proteins (HMG) 1 and 2 comprising two similar repeats, termed domains A and B, and an acidic C-terminal. In vitro, HMG1 and 2 can completely replace Ku protein as the DNA-binding regulatory component of DNA-PK. DNA-PKcs and Ku protein were separately purified from Raji nuclear extracts, and reconstituted into the DNA-PK holoenzyme in the presence of dsDNA. DNA-PKcs alone catalyzed DNA-dependent phosphorylation at a very low but significant level, and HMG1 and 2 markedly stimulate the phosphorylation of alpha-casein and a specific peptide substrate in a DNA-dependent manner. The HMG2-domains (A+B) polypeptide devoid of the C-terminal acidic region is more effective for DNA-PKcs stimulation than the full-length HMG2, and HMG2-domain A and B polypeptides. Antibodies to Ku protein inhibit the DNA-dependent phosphorylation activity of the DNA-PKcs:Ku protein complex, but not that of DNA-PKcs alone or when DNA-PKc is complexed with HMG1 or 2. These results demonstrate that either HMG1 or HMG2 can function as the DNA-binding regulatory component for DNA-PKcs in vitro, and imply that a conformational change of dsDNA, which is elicited by regulatory components, is important for the stimulation of DNA-PK activity of DNA-PKcs (Yumoto, 1998).

Mutation of HMG proteins

High mobility group 1 (HMG1) protein is an abundant component of all mammalian nuclei, and related proteins exist in all eukaryotes. HMG1 binds linear DNA with moderate affinity and no sequence specificity, but bends the double helix significantly on binding through the minor groove. It binds with high affinity to DNA that is already sharply bent, such as linker DNA at the entry and exit of nucleosomes; thus, it is considered a structural protein of chromatin. HMG1 is also recruited to DNA by interactions with proteins required for basal and regulated transcriptions and V(D)J recombination. Mice were generated harbouring deleted Hmg1. Hmg1-/- pups are born alive, but die within 24 hours due to hypoglycemia. Hmg1-deficient mice survive for several days if given glucose parenterally, then waste away with pleiotropic defects (but no alteration in the immune repertoire). Cell lines lacking Hmg1 grow normally, but the activation of gene expression by the glucocorticoid receptor (GR, encoded by the gene Grl1) is impaired. Thus, Hmg1 is not essential for the overall organization of chromatin in the cell nucleus, but is critical for proper transcriptional control by specific transcription factors (Calogero, 1999).

The immunosuppressive drugs FK506 and rapamycin bind to the cellular protein FKBP12, and the resulting FKBP12-drug complexes inhibit signal transduction. FKBP12 is a ubiquitous, highly conserved, abundant enzyme that catalyzes a rate-limiting step in protein folding: peptidyl-prolyl cis-trans isomerization. However, FKBP12 is dispensible for viability in both yeast and mice, and therefore does not play an essential role in protein folding. The functions of FKBP12 may involve interactions with a number of partner proteins, and a few proteins that interact with FKBP12 in the absence of FK506 or rapamycin have been identified, including the ryanodine receptor, aspartokinase, and the type II TGF-beta receptor; however, none of these are conserved from yeast to humans. To identify other targets and functions of FKBP12, a screen was carried out for mutations that are synthetically lethal with an FKBP12 mutation in yeast. Mutations in HMO1, which encodes a high mobility group 1/2 homolog, are synthetically lethal with mutations in the yeast FPR1 gene encoding FKBP12. Deltahmo1 and Deltafpr1 mutants share two phenotypes: an increased rate of plasmid loss and slow growth. In addition, Hmo1p and FKBP12 physically interact in FKBP12 affinity chromatography experiments, and two-hybrid experiments suggest that FKBP12 regulates Hmo1p-Hmo1p or Hmo1p-DNA interactions. Because HMG1/2 proteins are conserved from yeast to humans, these findings suggest that FKBP12-HMG1/2 interactions could represent the first conserved function of FKBP12 other than mediating FK506 and rapamycin actions (Dolinski, 1999).

DNA binding of HMG proteins

Lymphoid enhancer-binding factor 1 (LEF-1), a homolog of Drosophila Pangolin, is a regulatory high mobility group protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. The distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. Mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer. LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Recombinant LEF-1 activates HIV-1 transcription on chromatin-assembled DNA in vitro. The packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets-1 sites, further augments transcription in this system. A truncation mutant of LEF-1 (HMG-88) containing the HMG box but lacking the trans-activation domain, does not activate transcription from nucleosomal DNA, indicating that bending of DNA by the HMG domain is not sufficient to activate transcription in vitro. Therefore, transcription activation by LEF-1 in vitro appears to be a chromatin-dependent process that requires a functional trans-activation domain in addition to the HMG domain (Sheridan, 1995).

HMG1 is an evolutionarily highly conserved chromosomal protein consisting of two folded DNA-binding domains, A and B ('high mobility group (HMG) boxes'), and an acidic C-terminal domain. Several lines of evidence suggest that previously reported sequence-independent DNA bending and looping by HMG1 and its HMG box domains might be important for the proposed role of the protein in transcription and recombination. Ligase-mediated circularization assays were used to investigate the contribution of the individual A and B HMG1 box domains and of the linker region between A/B- and B/C-domains, which flanks the 'minimal' B-domain (residues 92-162), to the ability of the HMG1 protein (residues 1-215) to bend DNA. Neither the minimal B-domain nor the minimal B-domain with a 7-residue N-terminal extension (85TKKKFKD91) bends the DNA. The attachment of an extra 18-residue C-terminal additional extension (residues 163-180) to the minimal B-domain has only a small effect on the ability of the HMG box to bend DNA. In contrast, circularization assay with a B-domain having both 7-residue N-terminal and 18-residue C-terminal flanking sequences (residues 85-180) reveals a strong bending of the DNA, suggesting that both extensions are a prerequisite for efficient DNA bending by the B-domain. A single lysine residue (Lys90) in a short N-terminal sequence 90KD91 attached to the B-domain is sufficient for strong distortion of DNA by bending, provided that the B-domain is flanked by the 18-residue C-terminal flanking sequence. Although the DNA bending potential of HMG1 seems to be predominantly due to the B-domain flanked by basic sequences, covalent attachment of the A- and B-domains is necessary for efficient DNA flexure; the ability of the (A+B)-bidomain to bend DNA is further modulated in the native HMG1 protein by its acidic C-domain (Stros, 1998).

High-mobility-group protein 1 (HMG1) is a conserved chromosomal protein with two homologous DNA-binding HMG-box domains, A and B, linked by a short basic region to an acidic carboxy-terminal tail. NMR spectroscopy on the free didomain (AB) shows that the two HMG boxes do not interact. The didomain has a higher affinity for all DNA substrates tested than single HMG-box domains and has a significantly higher ability to distort DNA by bending and supercoiling. The interaction of the didomain with DNA is stabilized by the presence of the basic region (approximately 20 residues, 9 of which are Lys) that links the second HMG box to the acidic tail in intact HMG1; this may be, at least in part, why this region also enhances supercoiling of relaxed circular DNA by the didomain and circularization of short DNA fragments (in the presence of ligase). Competition assays suggest significantly different structure-specific preferences of single and tandem HMG boxes for four-way junction and supercoiled plasmid DNA. Binding to supercoiled DNA appears to be promoted by protein oligomerization, which is pronounced for the didomains. Electron microscopy suggests that the oligomers are globular aggregates, associated with DNA looping. One box versus two (or several) is likely to be an important determinant of the properties of (non-sequence specific) HMG-box proteins (Grasser, 1998).

The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous 'HMG-box' DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates) were examined. HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularization of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA (Lee, 2000).

HMG (high mobility group) 1 is a chromosomal protein with two homologous DNA-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG boxes, can recognize structural distortion of DNA, such as four-way DNA junctions (4WJs), that are very likely to have features common to their natural, yet unknown, cellular binding targets. HMG-1 can also bend/loop DNA and introduce negative supercoils in the presence of topoisomerase I in topologically closed DNAs. Results of gel shift assays demonstrate that mutation of Arg(97) within the extended N-terminal strand of the B domain significantly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Several basic amino acids of the extended N-terminal strand [Lys(96)/Arg(97)] and helix I [Arg(110)/Lys(114)] of the B domain participate in DNA binding and supercoiling. The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoiled DNA and 4WJs. It is concluded that the B domain of HMG-1 can tolerate substitutions of a number of amino acid residues without abolishing the structure-specific recognition of 4WJs, whereas mutations of most of these residues severely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive (Stros, 2000).

Chromatin architectural protein HMGB1 can bind with extremely high affinity (K(d) < 1 pM) to a novel DNA structure that forms a DNA loop maintained at its base by a hemicatenane (hcDNA). The loop of hcDNA contains a track of repetitive sequences derived from CA-microsatellites. Using a gel-retardation assay it is demonstrated that tumor-suppressor protein p53 can also bind to hcDNA. p53 is a crucial molecule protecting cells from malignant transformation by regulating cell-cycle progression, apoptosis, and DNA repair by activation or repression of transcription of its target genes by binding to specific p53 DNA-binding sites and/or certain types of DNA lesions or alternative DNA structures. The affinity of p53 for hcDNA (containing sequences with no resemblance to the p53 DNA consensus sequence) is >40-fold higher (K(d) approximately 0.5 nM) than that for its natural specific binding sites within its target genes (Mdm2 promoter). Binding of p53 to hcDNA remains detectable in the presence of up to approximately 4 orders of magnitude of mass excess of competitor linear DNA, suggesting a high specificity of the interaction. p53 displays a higher affinity for hcDNA than for DNA minicircles (lacking functional p53-specific binding sequence) with a size similar to that of the loop within the hcDNA, indicating that the extreme affinity of p53 for hcDNA is likely due to the binding of the protein to the hemicatenane. Although binding of p53 to hcDNA occurs in the absence of the nonspecific DNA-binding extreme carboxy-terminal regulatory domain (30-C, residues 363-393), the isolated 30-C domain (but not the sequence-specific p53 'core domain', residues 94-312) can also bind hcDNA. Only the full-length p53 can form stable ternary complexes with hcDNA and HMGB1. The possible biological relevance of p53 and HMGB1 binding to hemicatenanes is discussed (Stros, 2004).

HMG proteins, DNA bending and promoter function

HMG1 has been compared in four ways with the product of tryptic removal of its acidic C-terminal domain, termed HMG3, which contains two 'HMG-box' DNA-binding domains. (1) HMG3 has a higher affinity for DNA than HMG1. (2) Both HMG1 and HMG3 supercoil circular DNA in the presence of topoisomerase I. Supercoiling by HMG3 is the same at approximately 50 mM and approximately 150 mM ionic strength, as is its affinity for DNA, whereas supercoiling by HMG1 is less at 150 mM than at 50 mM ionic strength although its affinity for DNA is unchanged, showing that the acidic C-terminal tail represses supercoiling at the higher ionic strength. (3) Electron microscopy shows that HMG3 at a low protein:DNA input ratio, and HMG1 at a 6-fold higher ratio, cause looping of relaxed circular DNA at 150 mM ionic strength. Oligomeric protein 'beads' are apparent at the bases of the loops and at cross-overs of DNA duplexes. (4) HMG3 at high input ratios, but not HMG1, causes DNA compaction without distortion of the B-form. The two HMG-box domains of HMG1 are thus capable of manipulating DNA by looping, compaction and changes in topology. The acidic C-tail down-regulates these effects by modulation of the DNA-binding properties (Stros, 1994).

HMG-2 serves to activate the TFIID-TFIIA complex assembled on a promoter. HMG-2 is a factor necessary for activation in a defined transcription reaction in vitro containing RNA polymerase, and purified factors and a transcriptional activator. The activation process depends on TFIIA and on the presence of TAFs in the TFIID complex, and these generate a preinitiation complex from which TFIIB dissociates more slowly. The HMG box, as well as HMG-1 and HMG-2, binds DNA in the minor groove and bends the helix toward the major groove. Because the HMG box subdomain alone enables activation in the system, it is likely that these proteins function by stabilizing conformations of DNA bound to the basal factors and transcriptional activator. These critical conformations could involve looping of the activator to the basal complex or altering of the local topology of the DNA aroung the basal factors (Shykind, 1995).

The distal 3' enhancer of the T cell receptor alpha-chain (TCRalpha) gene directs the tissue- and stage-specific expression and V(D)J recombination of this gene locus. Using an in vitro system that efficiently reproduces TCRalpha enhancer activity, it can be shown that long-range promoter-enhancer regulation requires a T cell-specific repressor complex that operates on the proximal promoter and is sensitive to the DNA topology of the proximal promoter. In this system, the distal enhancer (regulated by ATF/CREB, LEF-1 [a homolog of Drosophila Pangolin], Ets-1 and GATA-3) functions to derepress the promoter on supercoiled, but not relaxed, templates. The TCRalpha promoter is inactivated by a repressor complex that contains the architectural protein HMG I/Y. HMG I/Y binds DNA through contacts in the minor groove, in contrast to both NF-kappaB (see Drosophila homologs Dorsal and Dif) and ATF-2, which both interact with the same sequences in the major groove. In the absence of this repressor complex, expression of the TCRalpha gene is completely independent of the 3' enhancer and DNA topology. The interaction of the T cell-restricted protein LEF-1 with the TCRalpha enhancer is required for promoter derepression. In this system, the TCRalpha enhancer increases the number of active promoters rather than the rate of transcription. Thus, long-range enhancers function in a distinct manner from promoters and provide the regulatory link between repressors, DNA topology, and gene activity (Bagga, 1997).

Tandemly repeated DNA sequences of (GGA:TCC)n are found in tracts up to 50 base pairs long, dispersed at thousands of sites throughout the genomes of eukaryotes. The formation of complexes paired between two DNAs containing such repeats occurs in vitro; enhancement of the pairing is facilitated by glutathione S-transferase fusion proteins of high mobility group protein 1 and histone H1 (see Drosophila Histone H1). This assembly depends on incubation time at 37 degrees C and concentrations of the proteins and DNA; the enhancement is inhibited by distamycin and actinomycin D interacting with DNA through the minor groove. The structure of the DNA-DNA complex is deduced by comparison of its mobility in gel electrophoresis with those of synthetic markers of heterotetramers. Three synthetic and genomic DNA fragments containing repeats that have different arrangements exhibit different efficiencies of DNA pairing, implying that the pairing is affected by the number of repeat units and the arrangement of repeats in a sequence. Intriguingly, pairing occurs between homologous fragments but not between heterologous DNAs among the three. These results suggest that the repeat-mediated DNA pairing plays a role in the organization of higher order architecture of chromatin and possibly chromosome segregation, requiring sequence-specific association events of DNA molecules (Mishima, 1997).

High mobility group (HMG) proteins are thought to facilitate assembly of higher order chromatin structure through modulation of DNA conformation. The bending of a 30-base pair DNA fragment induced by Chironomus HMG1 (cHMG1a), and HMGI (cHMGI) proteins, was examined. 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 is 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 has 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 is 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 (Heyduk, 1997).

The binding of p53 protein to DNA is stimulated by its interaction with covalent as well as noncovalent modifiers. A factor from HeLa nuclear extracts has been identified that activates p53 DNA binding. This factor was purified to homogeneity and identified as the high mobility group protein, HMG-1. HMG-1 belongs to a family of highly conserved chromatin-associated nucleoproteins that bend DNA and facilitate the binding of various transcription factors to their cognate DNA sequences. Recombinant His-tagged HMG-1 enhances p53 DNA binding in vitro, and HMG-1 and p53 can interact directly in vitro. Unexpectedly, HMG-1 also stimulates DNA binding by p53delta30, a carboxy-terminally deleted form of the protein that is considered to be constitutively active, suggesting that HMG-1 stimulates p53 by a mechanism that is distinct from other known activators of p53. HMG-1 can increase p53 and p53delta30-mediated transactivation in vivo. HMG-1 promotes the assembly of higher order p53 nucleoprotein structures. These data, along with the fact that HMG-1 is capable of bending DNA, suggest that HMG-1 may activate p53 DNA binding by a novel mechanism involving a structural change in the target DNA (Jayaraman, 1998).

HMGB-1/-2 are coregulatory proteins that facilitate the DNA binding and transcriptional activity of steroid receptor members of the nuclear receptor family of transcription factors. The influence and mechanism of action of HMGB-1/-2 (formerly known as HMG-1/-2) on estrogen receptor alpha (ERalpha) and ERbeta was investigated. Both ER subtypes are responsive to HMGB-1/-2 with respect to enhancement of receptor DNA binding affinity and transcriptional activity in cells. Responsiveness to HMGB-1/-2 is dependent on the C-terminal extension (CTE) region of the ER DNA binding domain (DBD) and correlated with a direct protein interaction between HMGB-1/-2 and the CTE. Thus the previously reported higher DNA binding affinity and transcription activity of ERalpha as compared with ERbeta is not due to a lack of ERbeta interaction with HMGB-1/-2. Using chimeric receptor DBDs, the higher intrinsic DNA binding affinity of ERalpha than ERbeta was shown to be due to a unique property of the ERalpha CTE, independent of HMGB-1/-2. The CTE of both ER subtypes was also shown to be required for interaction with ERE half-sites. These studies reveal the importance of the CTE and HMGB-1/-2 for ERalpha and ERbeta interaction with their cognate target DNAs (Melvin, 2004).

HMG proteins and the modification of chromatin structure

An experimental assay was developed to search for proteins capable of antagonizing histone H1-mediated general repression of transcription. T7 RNA polymerase templates containing an upstream scaffold-associated region (SAR) were highly selectively repressed by H1 relative to non-SAR control templates. This is due to the nucleation of H1 assembly into flanking DNA brought about by the numerous A-tracts (AT-rich sequences containing short homopolymeric runs of dA.dT base pairs) of the SAR. A partial, selective titration of these A-tracts by HMG protein HMG-I/Y leads to the complete derepression of transcription from the SAR template by inducing the redistribution of H1 onto non-SAR templates. SARs are associated with many highly transcribed regulated genes where they may serve to facilitate the HMG-I/Y-mediated displacement of histone H1 in chromatin. Indeed, HMG-I/Y was found to be strongly enriched in the H1-depleted subfraction that can be isolated from chromatin (Zhao, 1993).

Murine C-127 cell lines that overexpress high mobility group (HMG) proteins 1 and 2 were developed by transfection with cDNA sequences. The effects of these HMG proteins on the modulation of chromatin structure that accompanied transcription were examined. The levels of HMG1 mRNA and protein in cells overexpressing HMG1 protein were enhanced about 7- and 3-fold, respectively, in comparison with control cells, whereas those in cells overexpressing HMG2 protein were enhanced about 17- and 9-fold. The expression of reporter genes transfected into the cells was enhanced approximately 2-fold in cells overexpressing HMG1, but not HMG2, in comparison with those in control cells, irrespective of the sources of the genes and promoters. The minichromosome derived from the reporter plasmid in cells overexpressing HMG1 protein is more susceptible to micrococcal nuclease digestion than those in cells overexpressing HMG2 protein and control cells. The enhanced accessibility to micrococcal nuclease is not restricted to the expressing gene and promoter but involves the entire minichromosome, suggesting that the enhancement of gene expression results from changes in the condensation of the entire minichromosomal region by HMG1 protein. Minichromosomes in cells overexpressing HMG contain enhanced amounts of the respective HMG proteins and simultaneously reduced amounts of histone H1s. These results suggest that HMG1 and -2 proteins have different functions in the modulation of chromatin structure, and that HMG1 protein may sustain the structure of the respective gene to ensure that its activity as a template is expressed fully. These observations on the modulation of chromatin structure accompanying gene transcription in cells overexpressing HMG protein may provide important information on the function of these proteins (Ogawa, 1995).

HMG-I/Y belongs to a subclass of the high-mobility-group (HMG) proteins, a family of abundant low-molecular-weight mammalian chromosomal proteins. Three members of this subclass are known: HMG-I; HMG-Y, which differs by a deletion of 11 amino acids generated by alternative splicing of the HMG-I transcript; and HMG-IC, which is encoded by a distinct gene. HMG-I/Y and HMG-IC bind with high affinity to the minor groove of AT-rich DNA, an interaction mediated by a specific DNA-binding domain, the 'AT-hook', which is distinct from and unrelated to the 'HMG box' present in other HMG proteins. HMG-I, which contains three tandem repetitions of the AT-hook motif, was first isolated as a satellite-binding protein and was later shown to bind without specificity to any stretch of five to six dA/dT base pairs. It has a particularly high affinity in vitro for the AT-rich sequences found in SARs/MARs (scaffold/matrix-associated regions), from which it is able to displace histone H1, and could thus play a role in the modulation of chromatin structure and accessibility. Indeed, MATH-20, an artificial multi-AT-hook protein derived from HMG-I, has been shown to suppress the variegation of a white gene located close to AT-rich centromeric heterochromatin in Drosophila. This activity is consistent with the hypothesis that proteins related to HMG-I can effect changes in chromatin structure and, in this case, affect the functional properties of heterochromatin (Beaujean, 2000 and references therein).

In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. The nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect that is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. It is proposed that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state (Beaujean, 2000).

Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catalysed by ISWI are converted into relocation of DNA relative to a histone octamer. HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. Transient interaction of HMGB1 with nucleosomal linker DNA overlapping ISWI-binding sites enhances the ability of ACF to bind nucleosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity is inhibitory. These observations are consistent with a role for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling (Bonaldi, 2002).

Involvement of HMGB1 protein in human DNA mismatch repair

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. A novel component required for mismatch repair in human cells has been identified and characterized. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair (Yuan, 2004).

HMG protein and inflammatory responses

High Mobility Group 1 protein (HMGB1) is a chromatin component that, when leaked out by necrotic cells, triggers inflammation. HMGB1 can also be secreted by activated monocytes and macrophages, and functions as a late mediator of inflammation. Secretion of a nuclear protein requires a tightly controlled relocation program. HMGB1 shuttles actively, in all cells, between the nucleus and cytoplasm. Monocytes and macrophages acetylate HMGB1 extensively upon activation with lipopolysaccharide; moreover, forced hyperacetylation of HMGB1 in resting macrophages causes its relocalization to the cytosol. Cytosolic HMGB1 is then concentrated by default into secretory lysosomes, and secreted when monocytic cells receive an appropriate second signal (Bonaldi, 2003).

Despite significant advances in intensive care therapy and antibiotics, severe sepsis accounts for 9% of all deaths in the United States annually. The pathological sequelae of sepsis are characterized by a systemic inflammatory response, but experimental therapeutics that target specific early inflammatory mediators [tumor necrosis factor (TNF) and IL-1beta] have not proven efficacious in the clinic. High mobility group box 1 (HMGB1) has been identified as a late mediator of endotoxin-induced lethality that exhibits significantly delayed kinetics relative to TNF and IL-1beta. Serum HMGB1 levels are increased significantly in a standardized model of murine sepsis, beginning 18 h after surgical induction of peritonitis. Specific inhibition of HMGB1 activity [with either anti-HMGB1 antibody (600 microg per mouse) or the DNA-binding A box (600 microg per mouse)] beginning as late as 24 h after surgical induction of peritonitis significantly increases survival. Animals treated with either HMGB1 antagonist were protected against the development of organ injury, as evidenced by improved levels of serum creatinine and blood urea nitrogen. These observations demonstrate that specific inhibition of endogenous HMGB1 therapeutically reverses lethality of established sepsis indicating that HMGB1 inhibitors can be administered in a clinically relevant time frame (Yang, 2004).

High mobility group box 1 (HMGB1) protein, originally described as a DNA-binding protein that stabilizes nucleosomes and facilitates transcription, can also be released extracellularly during acute inflammatory responses. Exposure of neutrophils, monocytes, or macrophages to HMGB1 results in increased nuclear translocation of NF-kappaB and enhanced expression of proinflammatory cytokines. Although the receptor for advanced glycation end products (RAGE - glycation is an uncontrolled, non-enzymatic reaction of sugars with proteins) has been shown to interact with HMGB1, other putative HMGB1 receptors are known to exist but have not been characterized. In the present experiments, the role of RAGE, Toll-like receptor (TLR) 2, and TLR 4, as well as associated kinases, was investigated in HMGB1-induced cellular activation. Culture of neutrophils or macrophages with HMGB1 produce activation of NF-kappaB through TLR 4-independent mechanisms. Unlike lipopolysaccharide (LPS), which primarily increase the activity of IKKbeta, HMGB1 exposure results in activation of both IKKalpha and IKKbeta. Kinases and scaffolding proteins downstream of TLR 2 and TLR 4, but not TLR/interleukin-1 receptor (IL-1R)-independent kinases such as tumor necrosis factor receptor-associated factor 2, were involved in the enhancement of NF-kappaB-dependent transcription by HMGB1. Transfections with dominant negative constructs have demonstrated that TLR 2 and TLR 4 are both involved in HMGB1-induced activation of NF-kappaB. In contrast, RAGE plays only a minor role in macrophage activation by HMGB1. Interactions of HMGB1 with TLR 2 and TLR 4 may provide an explanation for the ability of HMGB1 to generate inflammatory responses that are similar to those initiated by LPS (Park, 2004)

The tumor suppressor microRNA let-7 represses the HMGA2 oncogene

HMGA2, a high-mobility group protein, is oncogenic in a variety of tumors, including benign mesenchymal tumors and lung cancers. Knockdown of Dicer in HeLa cells revealed that the HMGA2 gene is transcriptionally active, but its mRNA is destabilized in the cytoplasm through the microRNA (miRNA) pathway. HMGA2 is derepressed upon inhibition of let-7 in cells with high levels of the miRNA. Ectopic expression of let-7 reduces HMGA2 and cell proliferation in a lung cancer cell. The effect of let-7 on HMGA2 is dependent on multiple target sites in the 3' untranslated region (UTR), and the growth-suppressive effect of let-7 on lung cancer cells is rescued by overexpression of the HMGA2 ORF without a 3'UTR. These results provide a novel example of suppression of an oncogene by a tumor-suppressive miRNA and suggest that some tumors activate the oncogene through chromosomal translocations that eliminate the oncogene’s 3'UTR with the let-7 target sites (Lee, 2007).

Chromatin associated HMG proteins

Continued: see High mobility group protein D Evolutionary Homologs part 3/3 | back to part 1/3

High mobility group protein D: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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