HMG-box Domain Research Articles

LEF1 Is a Tumor Suppressor in T Cell Acute Lymphoblastic Leukemia

In an effort to further unravel the molecular pathogenesis of T cell acute lymphoblastic leukemia (T-ALL), we performed array CGH on diagnostic specimens from 47 pediatric patients with T-ALL. The LEF1 transcription factor is best known as a positive mediator of oncogenic β-catenin signaling, and it is required for the expression of MYC, Cyclin D1 and Survivin in some contexts, including some normal hematopoietic progenitors. Additionally, Lef1 has been shown to be required for the survival of murine Notch-dependent T cell lymphoma cells. We were thus surprised to find highly focal deletions of LEF1 in 10.6 % (n = 5 of 47) of primary T-ALL patient samples, with 3 cases harboring homozygous deletions and 2 harboring heterozygous deletions of this locus. These deletions involved no other known genes. We then sequenced this gene in 45 of these cases and found heterozygous mutations in 3 additional T-ALL samples, including 2 cases with frameshift mutations predicted to truncate the protein prior to its context-dependent activation and HMG box domains, and one case with an Asp85Asn substitution. Seven of the eight samples harboring LEF1 abnormalities also had mutations in the heterodimerization or PEST domains of NOTCH1. In an effort to characterize the biologic consequences of LEF1 loss in T-ALL, we took advantage of expression microarrays that were previously performed on most of these samples. LEF1 loss defines a novel T-ALL subtype characterized by arrest at the CD1-positive early cortical stage, and whose gene expression profile resembles that of HOX11-positive cases, although expression of HOX11 was generally low in these samples. Loss of LEF1 was mutually exclusive to overexpression of TAL1 and of the HOXA/MEIS1 cluster. Interestingly, LEF1-negative T-ALL cases were associated with increased expression of MYC, and gene set enrichment analysis identified a significant association between LEF1 loss and upregulation of MYC target genes. In addition to its role as a transcriptional activator in the setting of active WNT/β-catenin signaling, LEF1 can also act as a transcriptional repressor in some cellular contexts. Experiments are currently underway to establish the mechanism mediating the tumor suppressor activity of LEF1 in T-ALL.

Lymphoid enhancer factor interacts with GATA‐3 and controls its function in T helper type 2 cells

GATA-3 is the master transcription factor for T helper 2 (Th2) cell differentiation and is critical for the expression of Th2 cytokines. Little is known, however, about the nature of the functional molecular complexes of GATA-3. We identified a high-mobility group (HMG)-box type transcription factor, lymphoid enhancer factor 1 (LEF-1), in the GATA-3 complex present in Th2 cells using a Flag-calmodulin-binding peptide (CBP)-tag based proteomics method. The interaction between GATA-3 and LEF-1 was confirmed by co-immunoprecipitation experiments using LEF-1-introduced T-cell lineage TG40 cells. The HMG-box domain of LEF-1 and two zinc finger domains of GATA-3 were found to be important for the physical association. The introduction of LEF-1 into developing Th2 cells resulted in the suppression of Th2 cytokine production. The suppression was significantly lower in the cells into which a HMG-box-deleted LEF-1 mutant was introduced. Moreover, LEF-1 inhibited the binding activity of GATA-3 to the interleukin (IL)-5 promoter. These results suggest that LEF-1 is involved in the GATA-3 complex, while also regulating the GATA-3 function, such as the induction of Th2 cytokine expression via the inhibition of the DNA-binding activity of GATA-3.

Differences in the DNA-Binding Properties of the Hmg-Box Domains of HMG1 and the Sex-Determining Factor SRY

High-mobility-group protein 1 (HMG1) is an abundant, non-sequence-specific, chromosomal protein with two homologous, HMG-box, DNA-binding domains, A and B, and an acidic tail. The HMG-box motif also occurs, as a single copy, in some sequence-specific transcription factors, e.g. the sex-determining factor, SRY. We have investigated whether or not there are differences in the DNA-binding properties of the isolated A and B HMG-box domains of HMG1 and SRY and whether, in the case of A and B, there might also be differences due to different sequence contexts within the native protein. The basic regions that flank the HMG1 B box, giving B', enhance its DNA-binding, supercoiling and DNA-bending activities, and promote the self-association of the DNA-bound B-box. All the HMG-box domains bind with structure specificity to four-way junctions, but the structure selectivity is significantly greater for A and the SRY box than for the HMG1 B or B' domains, as judged by competition with excess plasmid DNA. The domains self-associate to different extents on supercoiled DNA and this may explain differences in the ability to discriminate between four-way junctions and supercoiled DNA. The HMG1 A, B and B' domains constrain negative superhelical turns in DNA, but the SRY HMG box does not. Only the full B domain (B') bends DNA in a ligase-mediated circularisation assay; the minimal B box, the A domain and the SRY box do not. Thus, despite a common global fold, the HMG box appears to have been adapted to various functions in different protein contexts.

Solution structure of the tandem HMG box domain from Human High mobility group protein B1

Solution structure of the tandem HMG box domain from Human High mobility group protein B1

HMG-box domain stimulation of RAG1/2 cleavage activity is metal ion dependent

BackgroundRAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, and Dai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction.ResultsWe show here that single HMG-box domains of HMGB1 stimulate RAG-mediated RSS cleavage in a concentration-dependent manner in the presence of Mn2+, but not Mg2+. Interestingly, the inability of a single HMG-box domain to stimulate RAG-mediated RSS cleavage in Mg2+ is overcome by the addition of partner RSS to promote synapsis. Furthermore, we show that mutant forms of HMGB1 which otherwise fail to stimulate RAG-mediated RSS cleavage in Mg2+ can be substantially rescued when Mg2+ is replaced with Mn2+.ConclusionThe conflicting data published previously in two different laboratories can be substantially explained by the choice of divalent metal ion and abundance of HMGB1 in the cleavage reaction. The observation that single HMG-box domains can promote RAG-mediated 23-RSS cleavage in Mg2+ in the presence, but not absence, of partner RSS suggests that synaptic complex assembly in vitro is associated with conformational changes that alter how the RAG and/or HMGB1 proteins bind and bend DNA in a manner that functionally replaces the role of one of the HMG-box domains in RAG-HMGB1 complexes assembled on a single RSS.

Proteomic screen defines the hepatocyte nuclear factor 1α-binding partners and identifies HMGB1 as a new cofactor of HNF1α

Hepatocyte nuclear factor (HNF)-1α is one of the liver-enriched transcription factors involved in many tissue-specific expressions of hepatic genes. The molecular mechanisms for determining HNF1α-mediated transactivation have not been explained fully. To identify unknown proteins that interact with HNF1α, we developed a co-IP-MS strategy to search HNF1α interactions, and high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel HNF1α-interacting protein. In vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation studies confirmed an interaction between HMGB1 and HNF1α. The protein–protein interaction was mediated through the HMG box domains of HMGB1 and the homeodomain of HNF1α. Furthermore, electrophoretic mobility shift assay and chromatin-immunoprecipitation assay demonstrated that HMGB1 was recruited to endogenous HNF1α-responsive promoters and enhanced HNF1α binding to its cognate DNA sequences. Moreover, luciferase reporter analyses showed that HMGB1 potentiated the transcriptional activities of HNF1α in cultured cells, and downregulation of HMGB1 by RNA interference specifically affected the HNF1α-dependent gene expression in HepG2 cell. Taken together, these findings raise the intriguing possibility that HMGB1 is a new cofactor of HNF1α and participates in HNF1α-mediated transcription regulation through protein–protein interaction.

High mobility group box (HMGB) proteins of Plasmodium falciparum: DNA binding proteins with pro-inflammatory activity

High mobility group box chromosomal protein 1 (HMGB1), known as an abundant, non-histone architectural chromosomal protein, is highly conserved across different species. Homologues of HMGB1 were identified and cloned from malaria parasite, Plasmodium falciparum. Sequence analyses showed that the P. falciparum HMGB1 (PfHMGB1) exhibits 45, 23 and 18%, while PfHMGB2 shares 42, 21 and 17% homology with Saccharomyces cerevisiae, human and mouse HMG box proteins respectively. Parasite PfHMGB1and PfHMGB2 proteins contain one HMG Box domain similar to B-Box of mammalian HMGB1. Electrophoretic Mobility Shift Assay (EMSA) showed that recombinant PfHMGB1 and PfHMGB2 bind to DNA. Immunofluorescence Assay using specific antibodies revealed that these proteins are expressed abundantly in the ring stage nuclei. Significant levels of PfHMGB1 and PfHMGB2 were also present in the parasite cytosol at trophozoite and schizont stages. Both, PfHMGB1 and PfHMGB2 were found to be potent inducers of pro-inflammatory cytokines such as TNFα from mouse peritoneal macrophages as analyzed by both reverse transcription PCR and by ELISA. These results suggest that secreted PfHMGB1 and PfHMGB2 may be responsible for eliciting/ triggering host inflammatory immune responses associated with malaria infection.

The Effects of Tcf-1 Gene Silence on Marrow CD4+ T Cells from Aplastic Anemia Patients.

As T-cell special expression gene, T cell factor 1 (Tcf-1) has an HMG-BOX domain be cognized as a transcription factor. We have found from our previous studies that Tcf-1 mRNA expression level in bone marrow (BM) CD4+ T cells from aplastic anemia (AA) patients is markedly higher than that of normal people. It is presumed that up-expression of Tcf-1 gene can be closely related to abnormal functions of BM CD4+ T cells from AA patients. Therefore in the current study, we discussed the effects of Tcf-1 gene silence on the BM CD4+ T cells from AA patients. Three shRNA expression vectors psiRNA1, psiRNA2 and psiRNA3 were constructed with psiRNA-hH1neo G2 plasmid. They were respectively transfected into Jurkat cells in which there was rather high expression level of Tcf-1 gene, then Tcf-1 mRNA of Jurkat cells was assayed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). All three plasmid-derived siRNAs could effectively reduce the expression level of Tcf-1 mRNA in transfected Jurkat cells, but the plasmid-derived siRNA psiRNA2 was found to be the most effective inhibitor of Tcf-1 expression. Next, the selected psiRNA2 were transfected into the BM CD4+ T cells from six AA patients. It was found that the expression level of Tcf-1 mRNA in BM CD4+ T cells from these patients was reduced by 45.0%. At the same time, the expression levels of c-myc and CD44 mRNA in BM CD4+ T cells from these AA patients were also decreased to different extents, 47.7% and 53.7%, respectively. These results indicate that Tcf-1 gene can regulate downstream target genes such as c-myc and CD44 to participate in over-proliferation and activation of BM CD4+ T cells from AA patients. It is likely worthwhile to carry out the in-depth study against Tcf-1 in AA therapy.

Chromosomal high mobility group (HMG) proteins of the HMGB-type occurring in the moss Physcomitrella patens

High mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures, which regulate DNA-dependent processes including transcription. Members of the HMGB family have been characterised from various mono-and dicot plants, but not from lower plant species. Here, we have identified three candidate HMGB proteins encoded in the genome of the moss Physcomitrella patens. The structurally similar HMGB2 and HMGB3 proteins display the typical overall structure of higher plant HMGB proteins consisting of a central HMG-box DNA-binding domain that is flanked by a basic N-terminal and an acidic C-terminal domain. The HMGB1 protein differs from higher plant HMGB proteins by having a very extensive N-terminal domain and by lacking the acidic C-terminal domain. Like higher plant HMGB proteins, HMGB3 localises to the cell nucleus, but HMGB1 is targeted to plastids. Analysis of the HMG-box domains of HMGB1 and HMGB3 by CD revealed that HMGB1box and the HMGB3box have an α-helical structure. While the HMGB3box interacts with DNA comparable to typical higher plant counterparts, the HMGB1box has only a low affinity for DNA. Cotransformation assays in Physcomitrella protoplasts demonstrated that expression of HMGB3 resulted in repression of reporter gene expression. In summary, our data show that functional HMGB-type proteins occur in Physcomitrella and most likely in other lower plant species.

HMGB1 interacts with human topoisomerase IIalpha and stimulates its catalytic activity.

DNA topoisomerase IIα (topo IIα) is an essential nuclear enzyme and its unique decatenation activity has been implicated in many aspects of chromosome dynamics such as chromosome replication and segregation during mitosis. Here we show that chromatin-associated protein HMGB1 (a member of the large family of HMG-box proteins with possible functions in DNA replication, transcription, recombination and DNA repair) promotes topo IIα-mediated catenation of circular DNA, relaxation of negatively supercoiled DNA and decatenation of kinetoplast DNA. HMGB1 interacts with topo IIα and this interaction, like the stimulation of the catalytic activity of the enzyme, requires both HMG-box domains of HMGB1. A mutant of HMGB1, which cannot change DNA topology stimulates DNA decatenation by topo IIα indistinguishably from the wild-type protein. Although HMGB1 stimulates ATP hydrolysis by topo IIα, the DNA cleavage is much more enhanced. The observed abilities of HMGB1 to interact with topo IIα and promote topo IIα binding to DNA suggest a mechanism by which HMGB1 stimulates the catalytic activity of the enzyme via enhancement of DNA cleavage.

The HMG-box: a versatile protein domain occurring in a wide variety of DNA-binding proteins.

The HMG-box domain of approximately 75 amino acid residues was originally identified as the domain that mediates the DNA-binding of chromatin-associated high-mobility group (HMG) proteins of the HMGB type. In the last few years, HMG-box domains have been found in various DNA-binding proteins including transcription factors and subunits of chromatin-remodeling complexes. HMG-box domains mediate either non-sequence-specific (e.g., HMGB-type proteins) or sequence-specific (e.g., transcription factors) DNA binding. Both types of HMG-box domains bind non-B-type DNA structures (bent, kinked and unwound) with high affinity. In addition, HMG-box domains are involved in a variety of protein-protein interactions. Here, we have examined the human and plant genomes for genes encoding HMG-box domains. Compared to plants, human cells contain a larger variety of HMG-box proteins. Whereas in humans transcription factors are the most divergent group of HMG-box proteins, in plants the chromosomal HMGB-type proteins are most variable.

Structure of human upstream binding factor HMG box 5 and site for binding of the cell-cycle regulatory factor TAF1

The fifth HMG-box domain in human upstream binding factor (hUBF) contributes to the synthesis of rRNA by RNA polymerase I (Pol I). The 2.0 A resolution crystal structure of this protein has been solved using the single-wavelength anomalous dispersion method (SAD). The crystal structure and the reported NMR structure have r.m.s. deviations of 2.18-3.03 A for the C(alpha) atoms. However, there are significant differences between the two structures, with displacements of up to 9.0 A. Compared with other HMG-box structures, the r.m.s. deviations for C(alpha) atoms between hUBF HMG box 5 and HMG domains from Drosophila melanogaster protein D and Rattus norvegicus HMG1 are 1.5 and 1.6 A, respectively. This indicates that the differences between the crystal and NMR structures of hUBF HMG box 5 are larger than those with its homologous structures. The differences between the two structures potentially reflect two states with different structures. The specific interactions between the hUBF HMG box 5 and the first bromodomain of TBP-associated factor 1 (TAF1) were studied by ultrasensitive differential scanning calorimetry and chemical shift perturbation. Based on these experimental data, possible sites in hUBF HMG box 5 that may interact with the first bromodomain of TAF1 were proposed.

Basic and Acidic Regions Flanking the HMG-Box Domain of Maize HMGB1 and HMGB5 Modulate the Stimulatory Effect on the DNA Binding of Transcription Factor Dof2

The chromatin-associated high-mobility group (HMG) proteins of the plant HMGB family are characterized by a central HMG-box domain that is flanked by a basic N-terminal and an acidic C-terminal domain. By functional interaction with certain transcription factors, HMGB proteins contribute to transcriptional regulation. Previous work has shown that the maize HMGB5 protein is markedly more efficient than other HMGB proteins in stimulating the binding of transcription factor Dof2 to DNA target sites. Here we examine the structural requirements that determine the particular efficiency of HMGB5. The HMG-box domains of HMGB1 and HMGB5 (which mediate the interaction with Dof2) promoted Dof2-DNA binding to a similar extent, indicating that the terminal domains modulate the interaction with Dof2. Analysis of full-length, truncated, and chimeric HMGB1/5 proteins revealed that the acidic C-terminal domains positively influence the stimulation of Dof2-DNA binding, while the basic N-terminal domains have a rather negative effect. In particular, the C-terminal domain of HMGB5 has a striking positive effect and may account for the efficient stimulation mediated by full-length HMGB5. Interestingly, recombinant HMGB protein variants that have a relatively low affinity for linear DNA (such as proteins lacking the basic N-terminal domain) efficiently assist Dof2-DNA binding.

Inhibitory Gli3 Activity Negatively Regulates Wnt/β-Catenin Signaling

The Hedgehog (Hh) and Wingless (Wnt) families of secreted signaling molecules have key roles in embryonic development and adult tissue homeostasis [1-3]. In the developing neural tube, Wnt and Shh, emanating from dorsal and ventral regions, respectively, have been proposed to govern the proliferation and survival of neural progenitors [4-10]. Surprisingly, Shh is required for the growth and survival of cells in both ventral and dorsal neural tube [11]. Here we demonstrate that inhibition of Shh signaling causes a reduction in Wnt-mediated transcriptional activation. This reduction requires Gli3. Assays in embryos and cell lines indicate that repressor forms of the Hh-regulated transcription factor, Gli3 (Gli3R), which are generated in the absence of Hh signaling, inhibit canonical Wnt signaling. Gli3R acts by antagonizing active forms of the Wnt transcriptional effector, beta-catenin. Consistent with this, Gli3R appears to physically interact with the carboxy-terminal domain of beta-catenin, a region that includes the transactivation domain. These data offer an explanation for the proliferative defects in Shh null embryos and suggest a novel mechanism for crosstalk between the Hh and Wnt pathways.

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.

Structure of a Complex of Tandem HMG Boxes and DNA

The high-mobility group protein HMGB1 contains two tandem DNA-binding HMG box domains, A and B, linked by a short flexible linker that allows the two domains to behave independently in the free protein. There is no structural information on how the linked domains and linker behave when bound to DNA, mainly due to the lack of any DNA-sequence preference of HMGB1. We report the structure determination, by NMR spectroscopy, of a well-defined complex of two tandem HMG boxes bound to a 16 bp oligonucleotide. The protein is an engineered version of the AB di-domain of HMGB1, in which the A box has been replaced by the HMG box of the sequence-specific transcription factor SRY, to give SRY.B. In the SRY.B/DNA complex, both HMG boxes bind in the minor groove and contribute to the overall DNA bending by intercalation of bulky hydrophobic residues between base-pairs; the bends reinforce each other, and the basic linker lies partly in the minor groove. As well as being the first structure of an HMG-box di-domain bound to DNA, this provides the first structure of the B domain of HMGB1 bound to DNA.

Isolation and Sequence Analysis of the Sox-1, -2, -3 Homologs in Trionyx sinensis and Alligator sinensis Having Temperature-Dependent Sex Determination

Members of the Sox gene family are characterized by an HMG-box that shows sequence similarity with that of the mouse testis-determining gene Sry. Using degenerate primers PCR, seven and eight HMG-box motifs of Sry-related genes were cloned and sequenced from genomic DNA of Trionyx sinensis (termed TS41-47) and Alligator sinensis (AS41-48) with TSD (temperature-dependent sex determination). Among 15 Sry-related genes, TS41, TS42, AS41, and AS42 shared 80, 72, 81, and 79% amino acid identity, respectively, with each HMG-box domain of the mouse Sox-1, -2, and -3 genes by Blast analysis. Molecular phylogenetic analysis showed that the clustering of TS41-42 and AS41-42 was distant to the clustering of the nonreptilian vertebrate Sox-1, -2, -3 homologs, including fish, amphibian, bird, and mammals. The amino acid identity among TS41-42, AS41-42, and the nonreptilian vertebrate Sox-1, -2, -3 homologs is lower than identities among the Sox-1, -2, -3 homologs, suggesting that the sequence changes in TS41-42 of Trionyx sinensis and AS41-42 of Alligator sinensis might have occurred after the diversification of amniotes.

Cloning the genes and DNA binding properties of High Mobility Group B1 (HMGB1) proteins from the human blood flukes Schistosoma mansoni and Schistosoma japonicum

The parasitic helminth Schistosoma mansoni contains three HMGB proteins, HMGB1, HMGB2 and HMGB3, of primary amino acid sequences highly similar to vertebrate proteins. In this report we describe the characterization of the HMGB1 proteins and their genes from S. mansoni and Schistosoma japonicum. The deduced amino acid sequences of HMGB1 proteins from both schistosome species are identical, and comprise 176 residues. The proteins contain the two evolutionarily highly conserved HMG-box domains, A and B, exhibiting 60% similarity to mammalian HMGB1. Unlike the human HMGB1 which contains an unbroken run of 30 glutamic or aspartic residues, the SmHMGB1 or SjHMGB1 proteins possess unusually short acidic C-terminal tails (5 acidic residues interrupted by 2 serines). Southern hybridization and DNA sequencing revealed a single copy HMGB1 gene, composed of 3 exons and two introns, in S. mansoni. The exon/intron boundaries are identical to those of the human HMGB1 gene, with the exception that the second exon of the SmHMGB1 gene which is not split into two exons as in the human HMGB1 gene. RNA blot analysis revealed that the SmHMGB1 gene is constitutively expressed in similar levels both in male and female worms. The single-sized mRNA for SmHMGB1 is consistent with the size derived from the cDNA. Although DNA binding properties of SmHMGB1 (or SjHMGB1) protein seem to be similar to those previously reported with human HMGB1, i.e., preferential binding to supercoiled DNA over linear DNA, specific recognition of DNA four-way junctions, DNA-induced supercoiling in the presence of topoisomerase I, and DNA bending, we have observed two important differences relative to those observed with the human HMGB1: (i) the inability of the isolated SmHMGB1 domain A to bend DNA (as revealed by T4 ligase-mediated circularization assay), and (ii) higher DNA supercoiling and bending potential of the SmHMGB1 protein as compared to its human counterpart. The latter finding may indicate that the long acidic C-tail of human HMGB1 has much stronger repressive role on DNA bending or DNA supercoiling by topoisomerase I at physiological ionic strength than the short C-tail of the SmHMGB1 protein. Considering the important role of HMGB1 in DNA replication, transcription, recombination, and in particularly, the mediation of inflammation responses in mammalian cells, further studies on schistosome HMGB proteins may provide valuable information related to schistosomiasis, where inflammation plays a critical role in this disease.

Ubc9 interacts with SOX4 and represses its transcriptional activity

SOX4 is a member of SOX transcriptional factor family that is crucial for many cellular processes. In this study, a yeast two-hybrid screening of human mammary cDNA library identified human ubiquitin-conjugating enzyme 9 (hUbc9) that interacted with SOX4. This interaction was confirmed by GST pull-down in vitro and co-immunoprecipitation assays in vivo. Deletion mapping demonstrated that HMG-box domain of SOX4 is required to mediate the interaction with Ubc9 in yeast. Furthermore, confocal microscopy showed that Ubc9 co-localized with SOX4 in the nucleus. Luciferase assays found that Ubc9 specifically repressed SOX4 transcriptional activity in 293T cells. We further demonstrated that Ubc9 could functionally repress the transcriptional activity of endogenous SOX4 induced by progesterone in T47D cells. The C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, did not abolish the ability to repress SOX4 activity. It shows that Ubc9 interacts with SOX4 and represses its transcriptional activity independent of its SUMO-1-conjugating activity.

The Arabidopsis Genome Encodes Structurally and Functionally Diverse HMGB-type Proteins

The high mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures, which regulate DNA-dependent processes including transcription and recombination. In addition to the previously identified HMGB1-HMGB6 proteins, the Arabidopsis genome encodes at least two other candidate family members (encoded by the loci At2g34450 and At5g23405) having the typical overall structure of a central domain displaying sequence similarity to HMG-box DNA binding domains, which is flanked by basic N-terminal and acidic C-terminal regions. Subcellular localisation experiments demonstrate that the At2g34450 protein is a nuclear protein, whereas the At5g23405 protein is found mainly in the cytoplasm. In line with this finding, At5g23405 displays specific interaction with the nuclear export receptor AtXPO1a. According to CD measurements, the HMG-box domains of both proteins have an alpha-helical structure. The HMG-box domain of At2g34450 interacts with linear DNA and binds structure-specifically to DNA minicircles, whereas the HMG-box domain of At5g23405 does not interact with DNA at all. In ligation experiments with short DNA fragments, the At2g34450 HMG-box domain can facilitate the formation of linear oligomers, but it does not promote the formation of DNA minicircles. Therefore, the At2g34450 protein shares several features with HMGB proteins, whereas the At5g23405 protein has different characteristics. Despite the presence of a region with similarity to the nucleosome-binding domain typical of HMGN proteins, At2g34450 does not bind nucleosome particles. In summary, our data demonstrate (i) that plant HMGB-type proteins are functionally variable and (ii) that it is difficult to predict HMG-box function solely based on sequence similarity.