High-mobility Group B Proteins Research Articles

Single and double box HMGB proteins differentially destabilize nucleosomes.

Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A–H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3–H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.

Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.

HMGB proteins involved in TOR signaling as general regulators of cell growth by controlling ribosome biogenesis.

The number of ribosomes and their activity need to be highly regulated because their function is crucial for the cell. Ribosome biogenesis is necessary for cell growth and proliferation in accordance with nutrient availability and other external and intracellular signals. High-mobility group B (HMGB) proteins are conserved from yeasts to human and are decisive in cellular fate. These proteins play critical functions, from the maintenance of chromatin structure, DNA repair, or transcriptional regulation, to facilitation of ribosome biogenesis. They are also involved in cancer and other pathologies. In this review, we summarize evidence of how HMGB proteins contribute to ribosome-biogenesis control, with special emphasis on a common nexus to the target of rapamycin (TOR) pathway, a signaling cascade essential for cell growth and proliferation from yeast to human. Perspectives in this field are also discussed.

Single and Double Box HMGB Proteins Differentially Destabilize Nucleosomes

Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A-H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.

High Mobility Group B Proteins, Their Partners, and Other Redox Sensors in Ovarian and Prostate Cancer.

Cancer cells try to avoid the overproduction of reactive oxygen species by metabolic rearrangements. These cells also develop specific strategies to increase ROS resistance and to express the enzymatic activities necessary for ROS detoxification. Oxidative stress produces DNA damage and also induces responses, which could help the cell to restore the initial equilibrium. But if this is not possible, oxidative stress finally activates signals that will lead to cell death. High mobility group B (HMGB) proteins have been previously related to the onset and progressions of cancers of different origins. The protein HMGB1 behaves as a redox sensor and its structural changes, which are conditioned by the oxidative environment, are associated with different functions of the protein. This review describes recent advances in the role of human HMGB proteins and other proteins interacting with them, in cancerous processes related to oxidative stress, with special reference to ovarian and prostate cancer. Their participation in the molecular mechanisms of resistance to cisplatin, a drug commonly used in chemotherapy, is also revised.

Cofractionation of HMGB proteins with histone dimers

An effective and flexible method is presented that can be used to investigate cofractionation of groups of nuclear proteins. The method was used to analyze chromatin-related proteins, of which high-mobility group B (HMGB) proteins consistently cofractionated by cation-exchange chromatography with the histone dimer (H2A–H2B). This led to the hypothesis that the two form a complex, further suggested by gel filtration, in which the HMGBs with core histones eluted as a defined high-molecular-weight peak. A necessary requirement for further studying protein interactions is that the constituents are of the highest possible purity and the pure histone dimers and tetramers used in this study were derived from pure histone octamers with their native marks. There is a growing interest in protein–protein interactions and an increasing focus on protein-interaction domains: most frequently, pull-down assays are used to examine these. The technology presented here can provide an effective system that complements pull-down assays.

Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A.

High-mobility group B (HMGB) proteins bind duplex DNA without sequence specificity, facilitating the formation of compact nucleoprotein structures by increasing the apparent flexibility of DNA through the introduction of DNA kinks. It has remained unclear whether HMGB binding and DNA kinking are simultaneous and whether the induced kink is rigid (static) or flexible. The detailed molecular mechanism of HMGB-induced DNA ‘softening’ is explored here by single-molecule fluorescence resonance energy transfer studies of single yeast Nhp6A (yNhp6A) proteins binding to short DNA duplexes. We show that the local effect of yNhp6A protein binding to DNA is consistent with formation of a single static kink that is short lived (lifetimes of a few seconds) under physiological buffer conditions. Within the time resolution of our experiments, this static kink occurs at the instant the protein binds to the DNA, and the DNA straightens at the instant the protein dissociates from the DNA. Our observations support a model in which HMGB proteins soften DNA through random dynamic binding and dissociation, accompanied by DNA kinking and straightening, respectively.

Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (∼10 s−1) are higher than those observed for wild-type proteins (∼0.1–1.0 s−1), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB–DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility.

Efficient purification of chromatin architectural proteins: histones, HMGB proteins and FKBP3 (FKBP25) immunophilin

A two-step process of high ionic strength lysis of chicken erythrocyte cell nuclei followed by cation-exchange chromatography has separated at very high yield all the histone and HMGB (high-mobility group B) nuclear proteins, except the less-soluble histone tetramers. Surprisingly high yields of the nuclear immunophilin FKBP3 (FKBP25) and Hsp70 (heat-shock protein 70) co-fractionate with HMGB1 and HMGB3. Furthermore, these proteins can be separated by anion-exchange chromatography. The purified nuclear proteins retain their native, post-translational modification (PTM) marks, including those associated with chromatin-fibre remodelling. These marks are intimately associated with the control of the cell cycle. The methods herein are therefore of value for targeting these and other nuclear proteins for future proteomic studies in healthy and diseased cells.

Involvement of High Mobility Group B Proteins in Cisplatin-Induced Cytotoxicity in Squamous Cell Carcinoma of Skin

Cis-diamminedichloroplatinum (II) (cisplatin) is a well-known anticancer drug with high potency and efficacy against various types of human cancers. Although it is widely accepted that the mechanism of cisplatin action is via apoptosis, there is enough evidence to support that cisplatin-induced cell death also occurs by other nonapoptotic pathways. Nonhistone, high mobility group (HMG) proteins are known to bind cisplatin-damaged DNA, and we studied their expression during cisplatin-induced cell death using immunohistochemistry, Western blot, and RT-PCR. Results show that the cell death is primarily apoptotic during initial stages of cisplatin treatment of skin tumors, and there is only marginal increase in high mobility group B (HMGB) levels, indicating that HMGB are still bound to nucleus. However, extended treatment of skin tumors with cisplatin caused necrosis and showed significantly increased levels of HMGB, which suggests that HMGB thus released from nuclei act as cytokine and trigger inflammatory response leading to necrosis. Present results clearly indicate a strong association between HMGB proteins and cisplatin-induced cell death that is dominantly apoptotic or necrotic depending on the duration of cisplatin exposure. Because of their important implication in the outcome of cancer chemotherapy, HMGB proteins can be interesting therapeutic targets.

Eukaryotic HMGB proteins as replacements for HU in E. coli repression loop formation.

DNA looping is important for gene repression and activation in Escherichia coli and is necessary for some kinds of gene regulation and recombination in eukaryotes. We are interested in sequence-nonspecific architectural DNA-binding proteins that alter the apparent flexibility of DNA by producing transient bends or kinks in DNA. The bacterial heat unstable (HU) and eukaryotic high-mobility group B (HMGB) proteins fall into this category. We have exploited a sensitive genetic assay of DNA looping in living E. coli cells to explore the extent to which HMGB proteins and derivatives can complement a DNA looping defect in E. coli lacking HU protein. Here, we show that derivatives of the yeast HMGB protein Nhp6A rescue DNA looping in E. coli lacking HU, in some cases facilitating looping to a greater extent than is observed in E. coli expressing normal levels of HU protein. Nhp6A-induced changes in the DNA length-dependence of repression efficiency suggest that Nhp6A alters DNA twist in vivo. In contrast, human HMGB2-box A derivatives did not rescue looping.

Characterization of Transgenic Arabidopsis Plants Overexpressing High Mobility Group B Proteins under High Salinity, Drought or Cold Stress

High mobility group B (HMGB) proteins found in the nuclei of higher eukaryotes play roles in various cellular processes such as replication, transcription and nucleosome assembly. The Arabidopsis thaliana genome contains eight genes encoding HMGB proteins, the functions of which remain largely unknown in the transcriptional regulation of plant stress responses. To understand better the functions of HMGB proteins in the responses of plants to environmental stimuli, we examined the effect of various abiotic stresses on germination and growth of transgenic Arabidopsis plants that overexpress a single isoform of HMGB. The expression of HMGB2, HMGB3 and HMGB4 was up-regulated by cold stress, whereas the expression of HMGB2 and HMGB3 was markedly down-regulated by drought or salt stress. Under salt or drought stress, the transgenic Arabidopsis plants that overexpress HMGB2 displayed retarded germination and subsequent growth compared with wild-type plants. Overexpression of HMGB4 had no impact on seed germination and seedling growth of the plants under the stress conditions tested. In contrast to no significant stress-related phenotypes of HMGB5-overexpressing plants, loss-of-function mutants of HMGB5 displayed retarded germination and subsequent growth compared with wild-type plants under stress conditions. Although transcript levels of various stress-responsive genes were not modulated by the expression of HMGB2, expression of several germination-responsive genes was modulated by HMGB2 under salt stress. Taken together, these results provide a novel basis for understanding the biological functions of HMGB protein family members that differently affect germination and seedling growth of Arabidopsis plants under various stress conditions.

Molecular cloning of a cDNA encoding a high mobility group protein in Cucumis sativus and its expression by abiotic stress treatments

A cDNA encoding a high mobility group B (HMGB) protein was isolated from Cucumis sativus and characterized with respect to its sequence, expression and responses to various abiotic stress treatments. The predicted polypeptide of 146 amino acid residues contains characteristic features of HMGB family proteins including the N-terminal basic region, one HMG-box and a stretch of acidic amino acid residues at the C-terminus. In vitro nucleic acid-binding assay revealed that the HMGB protein bound to both single-stranded DNA and double-stranded DNA. DNA gel blot analysis indicated that the HMGB gene is a single copy gene in cucumber genome. RNA gel blot analysis showed that the cucumber HMGB was more abundantly expressed in the roots than in shoots and leaves. Various abiotic stresses, including cold, drought and high salinity, down regulated markedly the expression of the HMGB in cucumber. The present report identifies a novel gene encoding HMGB protein in cucumber that shows a significant response to abiotic stress treatments.

Priming the nucleosome: a role for HMGB proteins?

The high-mobility-group B (HMGB) chromosomal proteins are characterized by the HMG box, a DNA-binding domain that both introduces a tight bend into DNA and binds preferentially to a variety of distorted DNA structures. The HMGB proteins seem to act primarily as architectural facilitators in the manipulation of nucleoprotein complexes; for example, in the assembly of complexes involved in recombination and transcription. Recent genetic and biochemical evidence suggests that these proteins can facilitate nucleosome remodelling. One mechanism by which HMGB proteins could prime the nucleosome for migration is to loosen the wrapped DNA and so enhance accessibility to chromatin-remodelling complexes and possibly also to transcription factors. By constraining a tight loop of untwisted DNA at the edge of a nucleosome, an HMGB protein could induce movements in the contacts between certain core histones that would result in an overall change in nucleosome structure.

Plant chromosomal HMGB proteins efficiently promote the bacterial site-specific beta-mediated recombination in vitro and in vivo.

In the presence of an accessory DNA bending protein, the bacterial site-specific beta recombinase catalyzes resolution and DNA inversion. Five different maize high mobility group B (HMGB) proteins were examined for their potential to facilitate beta recombination in vitro using DNA substrates with different intervening distances (73-913 bp) between two directly oriented recombination (six) sites. All analyzed HMGB proteins (HMGB1 to HMGB5) could promote beta recombination, but depending on the DNA substrate with different efficiencies. The HMGB1 protein displayed an activity comparable to that of the natural promoting protein Hbsu, whereas the other HMGB proteins were less effective. Phosphorylation of the HMGB1 protein resulted in an increased efficiency of HMGB1 to promote beta recombination. Analyses of DNA substrates with closely spaced six sites demonstrated that in the presence of HMGB1 the recombination rate was correlated to the distance between the six sites, but independent of the helical orientation of the six sites. Using a Bacillus subtilis strain defective in Hbsu, the coexpression of beta recombinase and HMGB1 (or a truncated HMGB1 derivative) revealed that a plant HMG-box domain protein is sufficient for assisting beta to catalyze recombination in vivo. Our results using beta recombination as a model system suggest that the various plant HMGB proteins (and their posttranslationally modified versions) have the potential of forming a repertoire of different DNA structures, which is compatible with the idea that the HMGB proteins can act as architectural factors in a variety of nucleoprotein structures.

Protein Kinase CK2 Differentially Phosphorylates Maize Chromosomal High Mobility Group B (HMGB) Proteins Modulating Their Stability and DNA Interactions

The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.