Interactions of the basic N-terminal and the acidic C-terminal domains of the maize chromosomal HMGB1 protein

Maize HMGB1 is a typical member of the family of plant chromosomal HMGB proteins, which have a central high-mobility group (HMG)-box DNA-binding domain that is flanked by a basic N-terminal region and a highly acidic C-terminal domain. The basic N-terminal domain positively influences various DNA interactions of the protein, while the acidic C-terminal domain has the opposite effect. Using DNA-cellulose binding and electrophoretic mobility shift assays, we demonstrate that the N-terminal basic domain binds DNA by itself, consistent with its positive effects on the DNA interactions of HMGB1. To examine whether the negative effect of the acidic C-terminal domain is brought about by interactions with the basic part of HMGB1 (N-terminal region, HMG-box domain), intramolecular cross-linking in combination with formic acid cleavage of the protein was used. These experiments revealed that the acidic C-terminal domain interacts with the basic N-terminal domain. The intramolecular interaction between the two oppositely charged termini of the protein is enhanced when serine residues in the acidic tail of HMGB1 are phosphorylated by protein kinase CK2, which can explain the negative effect of the phosphorylation on certain DNA interactions. In line with that, covalent cross-linking of the two terminal domains resulted in a reduced affinity of HMGB1 for linear DNA. Comparable to the finding with maize HMGB1, the basic N-terminal and the acidic C-terminal domains of the Arabidopsis HMGB1 and HMGB4 proteins interact, indicating that these intramolecular interactions, which can modulate HMGB protein function, generally occur in plant HMGB proteins.     

Both high mobility group (HMG)-boxes and the acidic tail of HMGB1 regulate recombination-activating gene (RAG)-mediated recombination signal synapsis and cleavage in vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.     

The evolution of High Mobility Group Box (HMGB) chromatin proteins in multicellular animals

Mammalian HMGB proteins are abundant chromatin components, and are characterized by the presence of 2 HMG-box domains and an acidic tail. HMG boxes are present in a large number of DNA-binding proteins, and HMGB chromatin proteins represent a small and specific subset of HMG-box proteins. The comparison of DNA sequences that code for HMG-box proteins suggests that the ancestral HMG box was coded by an intronless gene, which picked up one or more introns during its radiation. Canonical HMGB proteins are only present in multicellular animals, from sponges onwards, and appear to have arisen through the fusion of two different genes, each coding for one of the boxes. The organization of HMGB genes was very conserved during Metazoan evolution, with the only deviations appearing in Caenorhabditis and Dipteran (Drosophila and Anopheles) species.     

Interaction of maize chromatin-associated HMG proteins with mononucleosomes: role of core and linker histones

Two groups of plant chromatin-associated high mobility group (HMG) proteins, namely the HMGA family, typically containing four A/T-hook DNA-binding motifs, and the HMGB family, containing a single HMG-box DNA-binding domain, have been identified. We have examined the interaction of recombinant maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA in the presence of H1 differed from that observed in the absence of H1. HMGA and the HMGB proteins bound H1-containing nucleosome particles with similar affinity. The plant HMG proteins could also bind nucleosomes that were briefly treated with trypsin (removing the N-terminal domains of the core histones), suggesting that the histone N-termini are dispensable for HMG protein binding. In the presence of untreated nucleosomes and trypsinised nucleosomes, HMGB1 could be chemically crosslinked with a core histone, which indicates that the trypsin-resistant part of the histones within the nucleosome is the main interaction partner of HMGB1 rather than the histone N-termini. In conclusion, these results indicate that specific nucleosome binding of the plant HMGB proteins requires simultaneous DNA and histone contacts.     

Deciphering the role of the AT-rich interaction domain and the HMG-box domain of ARID-HMG proteins of Arabidopsis thaliana

ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (K_d?=?475?±?17.9 nM) compared to Arabidopsis HMGB1 protein (K_d?=?39.8?±?2.68 nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys⁸⁵ from the ARID domain and Arg¹⁹⁹ & Lys²⁰² from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.     

Localization and functional analysis of HmgB3p,a novel protein containing high-mobility-group-box domain from Tetrahymena thermophila

The high-mobility-group (HMG)-box domain represents a very versatile protein domain that mediates the DNA-binding of non-sequence-specific and sequence-specific proteins. HMG-box proteins are involved in various nuclear functions, including modulating chromatin structure and genomic stability. In this study, we identified the gene HMGB3 in Tetrahymena thermophila. The predicted HmgB3p contained a single HMG-box, an SK-rich-repeat domain and a neutral phosphorylated C-terminal. HMGB3 was expressed in the growth and starvation stages. Furthermore, HMGB3 showed a higher expression levels during the conjugation stage. HMGB3 knockout strains showed no obvious cytological defects, although initiation of HMGB3 knockout strain mating was delayed and maximum mating was decreased. HA-HmgB3p localized on the micronucleus (MIC) during the vegetative growth and starvation stages. Furthermore, HA-HmgB3p specially decorated the meiotic and mitotic functional MIC during the conjugation stage. Truncated HMGB3 lacking the HMG box domain disappeared from MICs and diffused in the cytoplasm. Overexpressed HmgB3p was abnormally maintained in newly developing macronuclei and affected the viability of progeny. Taken together, these results show that HmgB3p is a germline micronuclear-specific marker protein. It may bind to micronucleus-specific DNA sequences or structures and is likely to have some function specific to micronuclei of T. thermophila.     

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.     

Nature of full-length HMGB1 binding to cisplatin-modified DNA

HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.     

Rice HMGB1 protein recognizes DNA structures and bends DNA efficiently

We analyzed the DNA-binding and DNA-bending properties of recombinant HMGB1 proteins based on a rice HMGB1 cDNA. Electrophoretic mobility shift assay demonstrated that rice HMGB1 can bind synthetic four-way junction (4H) DNA and DNA minicircles efficiently but the binding to 4H can be completed out by HMGA and histone H1. Conformational changes were detected by circular dichroism analysis with 4H DNA bound to various concentrations of HMGB1 or its truncated forms. T4 ligase-mediated circularization assays with short DNA fragments of 123 bp showed that the protein is capable of increasing DNA flexibility. The 123-bp DNA formed closed circular monomers efficiently in its presence, similar to that in an earlier study on maize HMG. Additionally, our results show for the first time that the basic N-terminal domain enhances the affinity of the plant HMGB1 protein for 4H DNA, while the acidic C-terminal domain has the converse effects.     

Determinants of specific binding of HMGB1 protein to hemicatenated DNA loops

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.     

Purification and cDNA cloning of maize HMGd reveal a novel plant chromosomal HMG-box protein with sequence similarity to HMGa

We have purified the chromosomal high mobility group (HMG) protein HMGd from maize suspension culture cells, determined the N-terminal amino acid (aa) sequence, and isolated the corresponding cDNA. Sequence analysis showed that the cDNA encoded a protein of 126 aa residues with a theoretical mass of 14 104 Da. The protein contains an HMG-box DNA-binding domain and a short acidic C-terminal tail. HMGd is in approx. 65% of its residues identical to maize HMGa, whereas it is only approx. 46% identical to maize HMGcl/2. The differences to the previously reported HMG proteins in aa sequence, in overall charge and in protein size indicate that we have identified a third type of plant chromosomal HMG-box protein belonging to the HMG1 protein family. Immunoblot analysis with a HMGd antiserum reveals that HMGd is expressed in all tissues tested.