Diversification Pattern of the HMG and SOX Family Members During Evolution

From a database containing the published HMG protein sequences, we constructed an alignment of the HMG box functional domain based on sequence identity. Due to the large number of sequences (more than 250) and the short size of this domain, several data sets were used. This analysis reveals that the HMG box superfamily can be separated into two clearly defined subfamilies: (i) the SOX/MATA/TCF family, which clusters proteins able to bind to specific DNA sequences; and (ii) the HMG/UBF family, which clusters members which bind non specifically to DNA. The appearance and diversification of these subfamilies largely predate the split between the yeast and the metazoan lineages. Particular emphasis was placed on the analysis of the SOX subfamily. For the first time our analysis clearly identified the SOX subfamily as structured in six groups of genes named SOX5/6, SRY, SOX2/3, SOX14, SOX4/22, and SOX9/18. The validity of these gene clusters is confirmed by their functional characteristics and their sequences outside the HMG box. In sharp contrast, there are only a few robust branching patterns inside the UBF/HMG family, probably because of the much more ancient diversification of this family than the diversification of the SOX family. The only consistent groups that can be detected by our analysis are HMG box 1, vertebrate HMG box 2, insect SSRP, and plant HMG. The various UBF boxes cannot be clustered together and their diversification appears to be extremely ancient, probably before the appearance of metazoans. Received: 20 July 1998 / Accepted: 19 October 1998     

Structure of the HMG box motif in the B-domain of HMG1.

The conserved, abundant chromosomal protein HMG1 consists of two highly homologous, folded, basic DNA-binding domains, each of approximately 80 amino acid residues, and an acidic C-terminal tail. Each folded domain represents an 'HMG box', a sequence motif recently recognized in certain sequence-specific DNA-binding proteins and which also occurs in abundant HMG1-like proteins that bind to DNA without sequence specificity. The HMG box is defined by a set of highly conserved residues (most distinctively aromatic and basic) and appears to define a novel DNA-binding structural motif. We have expressed the HMG box region of the B-domain of rat HMG1 (residues 88-164 of the intact protein) in Escherichia coli and we describe here the determination of its structure by 2D 1H-NMR spectroscopy. There are three alpha-helices (residues 13-29, 34-48 and 50-74), which together account for approximately 75% of the total residues and contain many of the conserved basic and aromatic residues. Strikingly, the molecule is L-shaped, the angle of approximately 80 degrees between the two arms being defined by a cluster of conserved, predominantly aromatic, residues. The distinctive shape of the HMG box motif, which is distinct from hitherto characterized DNA-binding motifs, may be significant in relation to its recognition of four-way DNA junctions.     

Interactions between HMG boxes.

Many proteins consist of subdomains that can fold and function independently. We investigate here the interaction between the two high mobility group (HMG) box subdomains of the nuclear protein rHMG1. An HMG box is a conserved amino acid sequence of approximately 80 amino acids rich in basic, aromatic and proline side chains that is active in binding DNA in a sequence or structure-specific manner. In the case of HMG1, each box can bind structural DNA substrates including four-way junctions (4WJs) and branched or kinked DNA duplexes. Since proteins containing up to six HMG boxes are known, the question arises whether linking subdomains together influences the folding or function of individual boxes. In an effort to understand interactions between individual DNA-binding domains in HMG1, we created new fusion proteins: one is an inversion of the order of the AB di-domain in HMG1 (BA); in the second, we added a third A domain C-terminal to the AB di-domain (ABA). Pairs of boxes, AB or BA, behave similarly and are functionally active. By contrast, the ABA triple subdomain construct is partially unfolded and is less active than individual boxes or di-domains. Thus, long-range inter-domain effects can influence the activity of HMG boxes.     

Mutagenesis analysis of the interaction between the dorsal rel homology domain and HMG boxes of DSP1 protein

DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((253)KKRK(256)) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.     

The HMG domain of the ROX1 protein mediates repression of HEM13 through overlapping DNA binding and oligomerization functions.

The ROX1 gene of Saccharomyces cerevisiae encodes a protein required for the repression of genes expressed under anaerobic conditions. ROX1 belongs to a family of DNA binding proteins which contain the high mobility group motif (HMG domain). To ascertain whether the HMG domain of ROX1 is required for specific DNA binding we synthesized a series of ROX1 protein derivatives, either in vitro or in Escherichia coli as fusions to glutathione S-transferase (GST) protein, and tested them for their ability to bind to DNA. Both ROX1 proteins that were synthesized in vitro and GST-ROX1 fusion proteins containing the intact HMG domain were able to bind to specific target DNA sequences. In contrast, ROX1 proteins which contained deletions within the HMG domain were no longer capable of binding to DNA. The oligomerization of ROX1 in vitro was demonstrated using affinity-purified GST-ROXI protein and ROX1 labelled with [35S]methionine. Using various ROX1 protein derivatives we were able to demonstrate that the domain required for ROX1-ROX1 interaction resides within the N-terminal 100 amino acids which constitute the HMG domain. Therefore, the HMG domain is required for both DNA binding activity and oligomerization of ROX1.     

Structure-specific binding of the two tandem HMG boxes of HMG1 to four-way junction DNA is mediated by the A domain

We have investigated the nature of the "structure-specific" binding of the tandem A and B HMG boxes of high mobility group protein 1 (HMG1) to four-way junction DNA. AB didomain binding favours the open, planar form of the junction, as shown by reaction with potassium permanganate. Site-directed cleavage of the DNA by a 1, 10-phenanthroline-copper moiety attached to unique natural or engineered cysteine residues in the A or B domain shows that the two linked HMG boxes are not functionally equivalent in four-way junction binding. The A domain of the didomain binds to the centre of the junction, mediating structure-specific binding; the concave surface of the domain interacts with the widened minor groove at the centre, contacting one of the four strands of the junction, and the short arm comprising helices I and II and the connecting loop protrudes into the central hole. The B domain makes contacts along one of the arms, presumably stabilising the binding of the didomain through additional non-sequence-specific interactions. The isolated B domain can, however, bind to the centre of the junction. The preferential binding of the A domain of the AB didomain to the centre correlates with our previous finding of a higher preference of the isolated A domain than of the B domain for this structurally distinct DNA ligand. It is probably at least partly due to the higher positive surface potential in the DNA-binding region of the A domain (in particular to an array of positively charged side-chains suitably positioned to interact with the negatively charged phosphates surrounding the central hole of the junction) and partly to differences in residues corresponding to those that intercalate between bases in other HMG box/DNA complexes.     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.     

Identification of the core-histone-binding domains of HMG1 and HMG2

High mobility group (HMG) nonhistone chromosomal proteins are a group of abundant, conservative and highly charged nuclear proteins whose physiological role in chromatin is still unknown. To gain insight into the interactions of HMG1 and HMG2 with the fundamental components of chromatin we have introduced the methodology of photochemical crosslinking. This technique has allowed us to study the interaction of HMG1 and HMG2 with the core histones, in the form of an H2A X H2B dimer and an (H3 X H4)2 tetramer, for an effective time of crosslinking of less than 1 ms and under very mild conditions. This is achieved by using flash photolysis. With this procedure we found that both HMG1 and HMG2 interact with H2A X H2B and also with (H3 X H4)2. In the second case, they seem to do this through histone H3. To obtain more information about the interactions, we split HMG1 and HMG2 into their peptides using staphylococcal proteinase. The peptides obtained, which reflect the domain distribution of these proteins, were then used along with the histone oligomers to elucidate their interactions by means of photochemical crosslinking. Results obtained indicate that the domain of HMG1 and HMG2 involved in the interaction with H2A X H2B histones is the highly acidic C-terminal, whereas the N-terminal is involved in the interactions with (H3 X H4)2 histones. In all cases, the interactions found appear appreciably strong. Along with other data published in the literature, these proteins appear to have at least one binding site per domain for the chromatin components.     

中华鳖HMG1基因的克隆与序列分析

为了解中华鳖(Pelodiscus sinensis)HMG1(High mobility group 1)的基因结构,利用RT-PCR,从中华鳖肝脏组织的总RNA中,克隆并测序了中华鳖HMG1cDNA片段,结果表明,中华鳖HMG1基因的开放读码框(Open reading frame,ORF)长度为606 bp,编码202个氨基酸。中华鳖HMG1多肽链主要包含三个保守的区域:位于多肽链N端的HMG盒区1(第9—80个氨基酸之间);位于多肽链中心的HMG盒区2(第89—162个氨基酸之间);位于多肽链C端的富含酸性氨基酸区域(第163—202个氨基酸之间)。在2个HMG盒区范围内,中华鳖HMG1多肽链与红原鸡、人、虹鳟等物种的HMG1多肽链相比,氨基酸同源性依次为96.5%、74%和67%。排序比较显示,不同物种HMG1多肽链之间的富含酸性氨基酸区域的长度是不同的,暗示了HMG1多肽链富含酸性氨基酸区域的长度可能受到选择压力的影响,但这种选择压力没有使谷氨酸和天冬氨酸这两种酸性氨基酸之间区分开来。系统发生分析表明,脊椎动物HMG1基因的HMG盒区1和盒区2分别形成了2个亚族。本研究首次报道爬行动物的HMG1基因。       

Solution structure of the HMG protein NHP6A and its interaction with DNA reveals the structural determinants for non-sequence-specific binding

NHP6A is a chromatin-associated protein from Saccharomyces cerevisiae belonging to the HMG1/2 family of non-specific DNA binding proteins. NHP6A has only one HMG DNA binding domain and forms relatively stable complexes with DNA. We have determined the solution structure of NHP6A and constructed an NMR-based model structure of the DNA complex. The free NHP6A folds into an L-shaped three alpha-helix structure, and contains an unstructured 17 amino acid basic tail N-terminal to the HMG box. Intermolecular NOEs assigned between NHP6A and a 15 bp 13C,15N-labeled DNA duplex containing the SRY recognition sequence have positioned the NHP6A HMG domain onto the minor groove of the DNA at a site that is shifted by 1 bp and in reverse orientation from that found in the SRY-DNA complex. In the model structure of the NHP6A-DNA complex, the N-terminal basic tail is wrapped around the major groove in a manner mimicking the C-terminal tail of LEF1. The DNA in the complex is severely distorted and contains two adjacent kinks where side chains of methionine and phenylalanine that are important for bending are inserted. The NHP6A-DNA model structure provides insight into how this class of architectural DNA binding proteins may select preferential binding sites.     

The HMG1 ta(i)le

We have studied structural changes in DNA/protein complexes using the CD spectroscopy, upon the interaction of HMG1-domains with calf thymus DNA at different ionic strengths. HMG1 protein isolated from calf thymus and recombinant HMG1-(A+B) protein were used. Recombinant protein HMG1-(A+B) represents a rat HMG1 lacking C-terminal acidic tail. At low ionic strength (15 mM NaCl) we observed similar behavior of both proteins upon interaction with DNA. Despite this, at higher ionic strength (150 mM NaCl) their interaction with DNA leads to a completely different structure of the complexes. In the case of HMG1-(A+B)/DNA complexes we observed the appearance of DNA fractions possessing very high optical activity. This could be a result of formation of the highly-ordered DNA structures modulated by the interaction with HMG1-domains. Thus the comparison studies of HMG1 and HMG1-(A+B) interaction with DNA show that negatively charged C-terminal tail of HMG1 modulates interaction of the protein with DNA. The striking difference of the behaviour of these two systems allows us to explain the functional role of multiple HMG1 domains in some regulatory and architectural proteins.     

高迁移率族蛋白成员HMG20a/b的研究进展

高迁移率族(HMG)蛋白是一类广泛存在的非组蛋白型染色体蛋白,能通过诱导染色质结构的变化影响DNA表达。HMG20a和HMG20b是一对高度同源的HMG家族蛋白,均含有一个结构保守的HMG-box结构域和一个coiled-coil结构域,在生物体内广泛表达。HMG20a/b在细胞核内参与组蛋白去甲基酶复合物LSD1-Co REST的形成及一系列与细胞分裂分化相关的生理进程,如神经细胞核红细胞的分化、细胞质分裂以及EMT过程。研究发现,HMG20a/b一些功能的发挥是通过LSD1-Co REST复合物来实现的;在神经分化过程中,HMG20a、HMG20b具有相互拮抗的作用;而HMG20a促进EMT过程反映它很可能是一个促癌因子。本文对HMG20a/b的结构和体内分布及生物学功能进行综述。     

Effect of HMG protein 17 on the thermal stability of control and acetylated HeLa oligonucleosomes.

Many studies have implicated histone acetylation and HMG proteins 14 and 17 in the structure of active chromatin. Studies of the binding of HMG 14 and 17 to chromatin core particles have shown that there are two binding sites for HMG 14 or 17 located within 20-25 bp of the DNA ends of the core particles [13-15]. Such binding sites may result from the free DNA ends in the core particle being available for the binding of HMG 14 and 17. We have studied the effects of the binding of HMG 17 on the thermal denaturation of DNA in mono, di and trinucleosomes. In each case the binding of 1 HMG 17 molecule per nucleosome reduces the DNA premelt region by 50%, while the binding of 2 HMG 17 molecules per nucleosome abolishes the premelt region. From this it is concluded that there are two HMG 17 binding sites per nucleosome which are located between the entry and exit points to the nucleosome and the strongly complexed central DNA region. Highly acetylated mono, di and trinucleosomes have been isolated from butyrate treated HeLa S3 cells. For this series of acetylated oligonucleosomes, it has been found that there are also two HMG 17 binding sites per acetylated nucleosome.