High Mobility Group Boxes Research Articles

Gonadal Transcriptomic Analysis Reveals Novel Sex-Related Genes in Bactrocera dorsalis.

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is one of the most devastating agricultural pests worldwide due to its high reproductive and invasive abilities. The elucidation of its gonadal developmental characteristics and the identification of sex-related genes will provide a useful genetic basis for reproductive-based pest control. Here, the gonadal transcriptome of B. dorsalis was sequenced, and novel gonad-specific expressed genes were analyzed. A total of 1338, 336, 35, and 479 differentially expressed genes (DEGs) were found in the testis (TE), ovary (OV), female accessory gland (FAG), and male accessory gland (MAG), respectively. Furthermore, 463 highly expressed gonad-specific genes were identified, with the TE having the highest number of specific highly expressed genes, at 402, followed by 51 in the OV, 9 in the MAG, and only 1 in the FAG. Strikingly, approximately half of highly expressed gonad-specific genes were uncharacterized. Then, it was found that 35, 17, 3, 2, and 1 of 202 uncharacterized highly expressed TE-specific genes encoded proteins that contained transmembrane domains, signal peptides, high-mobility group boxes, the zinc finger domain, and the BTB/POZ domain, respectively. Interestingly, approximately 40% of uncharacterized highly expressed gonad-specific genes encoding proteins were not predicted to possess functional motifs or domains. Finally, the spatiotemporal expression and sequence characterization of six novel highly expressed gonad-specific genes were analyzed. Altogether, our findings provide a valuable dataset for future functional analyses of sex-related genes and potential target sites for pest control.

Structure and dynamics of the mitochondrial DNA-compaction factor Abf2 from S. cerevisiae

Mitochondria are essential organelles that produce most of the energy via the oxidative phosphorylation (OXPHOS) system in all eukaryotic cells. Several essential subunits of the OXPHOS system are encoded by the mitochondrial genome (mtDNA) despite its small size. Defects in mtDNA maintenance and expression can lead to severe OXPHOS deficiencies and are amongst the most common causes of mitochondrial disease. The mtDNA is packaged as nucleoprotein structures, referred to as nucleoids. The conserved mitochondrial proteins, ARS-binding factor 2 (Abf2) in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) and mitochondrial transcription factor A (TFAM) in mammals, are nucleoid associated proteins (NAPs) acting as condensing factors needed for packaging and maintenance of the mtDNA. Interestingly, gene knockout of Abf2 leads, in yeast, to the loss of mtDNA and respiratory growth, providing evidence for its crucial role. On a structural level, the condensing factors usually contain two DNA binding domains called high-mobility group boxes (HMG boxes). The co-operating mechanical activities of these domains are crucial in establishing the nucleoid architecture by bending the DNA strands. Here we used advanced solution NMR spectroscopy methods to characterize the dynamical properties of Abf2 together with its interaction with DNA. We find that the two HMG-domains react notably different to external cues like temperature and salt, indicating distinct functional properties. Biophysical characterizations show the pronounced difference of these domains upon DNA-binding, suggesting a refined picture of the Abf2 functional cycle.

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity.

Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP, de novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

DNA bending by the chromosomal protein HMG1 and its high mobility group box domains. Effect of flanking sequences.

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. We have used ligase-mediated circularization assays 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 flank 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) bent the DNA. The attachment of an extra 18-residue C-terminal additional extension (residues 163-180) to the minimal B-domain had only a small effect on the ability of the HMG box to bend DNA. On the other hand, circularization assay with a B-domain having both 7-residue N-terminal and 18-residue C-terminal flanking sequences (residues 85-180) revealed a strong bending of the DNA, suggesting that both extensions are a prerequisite for efficient DNA bending by the B-domain. We have also shown that 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 and the ability of the (A+B)-bidomain to bend DNA is further modulated in the native HMG1 protein by its acidic C-domain.

Internal Associations of the Acidic Region of Upstream Binding Factor Control Its Nucleolar Localization.

Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization.

Attenuation of thioacetamide-induced hepatocellular injury by short-term repeated injections associated with down-regulation of metabolic enzymes and relationship with MHC class II-presenting cells

The liver is the primary organ participating in the metabolism of xenobiotics and is therefore an important target in the safety assessment of drugs, chemicals and environmental toxins. Drug-induced liver injury (DILI) has recently become widely recognized in human medicine as an adverse event. The progression of DILI often involves “damage-associated molecular patterns” (DAMPs) of gene and protein expression such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (Hsp). DAMPs are released from injured or necrotic cells and are bound to Toll-like receptors (TLRs) and modulate inflammatory reactions. Previously, in thioacetamide (TAA; 300mg/kg body weight, single injection)-induced rat liver, we demonstrated that the expressions of DAMPs, TLR4 and major histocompatibility complex (MHC) class II were simultaneously increased, accompanied with progression of hepatocellular injury and inflammation. Here we investigated the association of DILI and DAMPs, TLRs and MHC class II by using rat livers repeated injections with TAA (100mg/kg body weight, once, three times). Two days after TAA single injection, centrilobular hepatocellular necrosis with infiltration of mononuclear cells was observed, being paralleled with increase in serum levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP). However, two days after duplicate and triplicate injections, only mild degenerative change of hepatocytes and slight infiltration of mononuclear cells were seen in the affected centrilobular area. Serum levels of AST, ALT and ALP were also decreased to the same levels of control. mRNA expressions of DAMPs (HMGBs, S100A4 and Hsp 70-2), TLR4 and MHC class II tended to be increased only on single injection, although the number of MHC class II-positive cells in the centrilobular area was still increased on each examination point. The analysis of enzymes (CYP2E1 and Flavin monooxygenase (FMO) 3), which metabolize TAA in hepatocytes, showed a significant decrease in FMO3 on the duplicate and triplicate injections. Autophagy and regulatory T cells were not significantly changed for the attenuation of hepatocyte injury. Collectively, these results suggest that hepatocytes may adapt accumulation of the toxicant by changing their enzyme functions; furthermore, MHC class II cells, which still showed increased number in the duplicate and triplicate injections, may be related with protection from the toxicant.

The kinetics of damage-associated molecular patterns (DAMPs) and toll-like receptors during thioacetamide-induced acute liver injury in rats

Drug-induced liver injury (DILI) is a common problem in human medicine and it is a major reason to withdraw marketed drugs. However, the mechanism of DILI is still less known. Damage-associated molecular patterns (DAMPs), such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (HSPs), are released from injured or necrotic cells, bind to toll-like receptors (TLRs) and modulate inflammatory reactions. Here we investigated the kinetics of DAMPs, TLRs and MHC class II in a rat model of DILI with thioacetamide (TAA). After TAA administration, extensive necrosis was observed on days 1 and 2, followed by infiltration of inflammatory cells on day 3. The levels of serum liver enzymes also peaked on day 1. Expression of HMGB-1, -2 and S100A4 peaked on day 2. TLR-4 was up-regulated on day 3. The number of MHC class II-positive macrophages increased until day 2. These results suggest that HMGB-1, -2 and S100A4 are associated with hepatocellular necrosis and that DAMPs may activate TLR-4 and MHC class II during TAA-induced liver injury. Our data would contribute to the elucidation of the mechanism of DILI.

“Protein Un‐Design”: Evolutionary Origins of Native DNA‐Binding Architectures in Unfolded Polypeptides

We seek to test the hypothesis that the evolutionary origins of globular proteins, however exquisite in their structural organization, lie within the conformational repertoire of unstructured polypeptides. A model is provided by the high‐mobility‐group (HMG) box, an L‐shaped a‐helical motif of sharp DNA bending that represents a primordial innovation at the root of Eukarya. Whereas the modern HMG box exhibits conserved architectural features (such as an “aromatic buttress” underlying its angular protein surface; Racca, J., et al. J. Biol. Chem. E‐pub (2014)), we demonstrate that natively unfolded variants with defective buttresses can, as a seeming paradox, exhibit native DNA binding and DNA bending. Canonical structure is regained on specific DNA binding as inferred from CD and 1H‐15N NMR. Coupled DNA‐dependent protein folding and DNA bending suggest that primordial boxes could readily have evolved from unstructured precursors, driven by selection for architectural gene regulation. Functional “proof of principle” is provided by unfolded variants of human testis‐determining factor SRY. In a rat embryonic gonadal‐ridge cell line such variant HMG boxes are able to regulate the principal target gene in the nascent testis (Sox9), activating a male transcriptional program. We envisage that progressive stabilization of nascent folds represents a fundamental mechanism for the rapid evolution of autonomous protein structures, yielding the modern landscape of DNA‐binding architectures. In this view extant unstructured DNA‐ and RNA‐binding motifs represent relics of the transition between RNA and protein worlds.Acknowledgements. We thank L. Labeots, A. Jansco‐Radek, J. Radek, and G. Chan for their participation in the early stages of this work.

HMGB1-Facilitated p53 DNA Binding Occurs via HMG-Box/p53 Transactivation Domain Interaction, Regulated by the Acidic Tail

Facilitated binding of p53 to DNA by high mobility group B1 (HMGB1) may involve interaction between the N-terminal region of p53 and the high mobility group (HMG) boxes, as well as HMG-induced bending of the DNA. Intramolecular shielding of the boxes by the HMGB1 acidic tail results in an unstable complex with p53 until the tail is truncated to half its length, at which point the A box, proposed to be the preferred binding site for p53(1-93), is exposed, leaving the B box to bind and bend DNA. The A box interacts with residues 38-61 (TAD2) of the p53 transactivation domain. Residues 19-26 (TAD1) bind weakly, but only in the context of p53(1-93) and not as a free TAD1 peptide. We have solved the structure of the A-box/p53(1-93) complex by nuclear magnetic resonance spectroscopy. The incipient amphipathic helix in TAD2 recognizes the concave DNA-binding face of the A box and may be acting as a single-stranded DNA mimic.

Cytosolic DNA-Activated Human Dendritic Cells Are Potent Activators of the Adaptive Immune Response

Recent studies in cell lines and genetically engineered mice have demonstrated that cytosolic dsDNA could activate dendritic cells (DCs) to become effector APCs. Recognition of DNA might be a major factor in antimicrobial immune responses against cytosolic pathogens and also in human autoimmune diseases such as systemic lupus erythematosus. However, the role of cytosolic dsDNA in human DC activation and its effects on effector T and B cells are still elusive. In this study, we demonstrate that intracellular dsDNA is a potent activator of human monocyte-derived DCs as well as primary DCs. Activation by dsDNA depends on NF-κB activation, partially on the adaptor molecule IFN-promoter stimulator-1 and the novel cytosolic dsDNA receptor IFI16, but not on the previously recognized dsDNA sentinels absent in melanoma 2, DNA-dependent activator of IFN regulatory factor 3, RNA polymerase III, or high-mobility group boxes. More importantly, we report for the first time, to our knowledge, that human dsDNA-activated DCs, rather than LPS- or inflammatory cytokine mixture-activated DCs, represent the most potent inducers of naive CD4(+) T cells to promote Th1-type cytokine production and generate CD4(+) and CD8(+) cytotoxic T cells. dsDNA-DCs, but not LPS- or mixture-activated DCs, induce B cells to produce complement-fixing IgG1 and IgG3 Abs. We propose that cytosolic dsDNA represents a novel, more effective approach to generate DCs to enhance vaccine effectiveness in reprogramming the adaptive immune system to eradicate infectious agents, autoimmunity, allergy, and cancer.

Unexpected mobility of plant chromatin-associated HMGB proteins

High mobility group (HMG) proteins of the HMGB family containing a highly conserved HMG box are chromatin-associated proteins that interact with DNA and nucleosomes and catalyze changes in DNA topology, thereby facilitating important DNA-dependent processes. The genome of Arabidopsis thaliana encodes 15 different HMG-box proteins that are further subdivided into four groups: HMGB-type proteins, ARID-HMG proteins, 3xHMG proteins that contain three HMG boxes and the structure-specific recognition protein 1 (SSRP1). Typically, HMGB proteins are localized exclusively to the nucleus, like Arabidopsis HMGB1 and B5. However, these Arabidopsis HMGB proteins showed a very high mobility within the nuclear compartment. Recent studies revealed that Arabidopsis HMGB2/3 and B4 proteins are predominantly nuclear but also exist in the cytoplasm, suggesting an as yet unknown cytoplasmic function of these chromosomal HMG proteins.

Novel subfamily of mitochondrial HMG box-containing proteins: functional analysis of Gcf1p from Candida albicans

Mitochondria of eukaryotic organisms contain populations of DNA molecules that are packed into higher-order structures called mitochondrial nucleoids (mt-nucleoids). In Saccharomyces cerevisiae, the compaction of mitochondrial DNA (mtDNA) into mt-nucleoids is mediated primarily by the high-mobility group (HMG) box-containing protein Abf2, which is an important player in stabilization and metabolism of mtDNA. Although it is evident that analogous proteins must exist in other yeast species, an apparently fast divergence rate has precluded their identification, characterization and comparative analysis. Using in silico analysis of the complete genome sequence of the pathogenic yeast Candida albicans we predicted that the ORF 19.400/19.8030 assigned as GCF1 encodes a putative mitochondrial HMG box-containing protein. In contrast to Abf2p, which contains two HMG boxes, Gcf1p contains only one C-terminal HMG box. In addition, it contains one putative coiled-coil domain with a potential role in protein dimerization. Fluorescence microscopy analysis of a C-terminally tagged Gcf1p with green fluorescent protein (GFP) revealed its mitochondrial localization in both heterologous (S. cerevisiae) and native (C. albicans) hosts. Biochemical analyses of DNA-binding properties indicate that Gcf1p is, similarly to Abf2p, a non-specific DNA-binding protein. To analyse the role of Gcf1p in mtDNA metabolism, we constructed strains lacking one functional allele of the GCF1 gene and carrying one GCF1 allele under the control of the MET3 promoter. Under repressible conditions this strain exhibited a more than 3000-fold decrease in levels of GCF1 mRNA, which was correlated with a substantial decrease in the number of mtDNA copies as well as recombination intermediates. The dramatic effect of reduced levels of Gcf1p on mtDNA metabolism indicates that the protein is involved in essential molecular transactions that relate to the mitochondrial genome.

Microsporidia Evolved from Ancestral Sexual Fungi

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

SRY-related (Sox) genes in the genome of European Atlantic sturgeon (Acipenser sturio)

The Sox-gene family represents an ancient group of transcription factors involved in numerous developmental processes and sex determination in vertebrates. SOX proteins are characterized by a conserved high mobility group (HMG)-box domain, which is responsible for DNA binding and bending. We studied Sox genes in sturgeon, one of the most primitive groups of fishes characterized by a high chromosome number. Male and female genomes were screened for Sox genes using highly degenerate primers that amplified a broad range of HMG boxes. A total of 102 clones, representing 22 different sequences coding for 8 Sox genes, was detected and classified according to their orthologues. Sox2, Sox3, Sox4, Sox9, Sox11, Sox17, Sox19, and Sox21 were found in sturgeon; these genes represent Sox groups B, C, E, and F. In a phylogenetic analysis (neighbor-joining, maximum likelihood, maximum parsimony), these genes clustered with their mouse orthologues. In the case of Sox4, Sox17, and Sox21, we found evidence of gene duplication.

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.

Functional Domains of Chicken Mitochondrial Transcription Factor A for the Maintenance of Mitochondrial DNA Copy Number in Lymphoma Cell Line DT40

Nuclear and mitochondrial (mt) forms of chicken mt transcription factor A (c-TFAM) generated by alternative splicing of a gene (c-tfam) were cloned. c-tfam mapped at 6q1.1-q1.2 has similar exon/intron organization as mouse tfam except that the first exons encoding the nuclear and the mt form-specific sequences were positioned oppositely. When cDNA encoding the nuclear form was transiently expressed in chicken lymphoma DT40 cells after tagging at the C terminus with c-Myc, the product was localized into nucleus, whereas the only endogenous mt form of DT40 cells was immunostained exclusively within mitochondria. c-TFAM is most similar to Xenopus (xl-) TFAM in having extended C-terminal regions in addition to two high mobility group (HMG) boxes, a linker region between them, and a C-terminal tail, also found in human and mouse TFAM. Similarities between c- and xl-TFAM are higher in linker and C-terminal regions than in HMG boxes. Disruption of both tfam alleles in DT40 cells prevented proliferation. The tfam+/tfam- cells showed a 50 and 40-60% reduction of mtDNA and its transcripts, respectively. Expression of exogenous wild type c-tfam cDNA in the tfam+/tfam- cells increased mtDNA up to 4-fold in a dose-dependent manner, whereas its transcripts increased only marginally. A deletion mutant lacking the first HMG box lost this activity, whereas only marginal reduction of the activity was observed in a deletion mutant at the second HMG box. Despite the essential role of the C-terminal tail in mtDNA transcription demonstrated in vitro, deletion of c-TFAM at this region reduced the activity of maintenance of the mtDNA level only by 50%. A series of deletion mutant at the tail region suggested stimulatory and suppressive sequences in this region for the maintenance of mtDNA level.

Identification of Novel Domains within Sox-2 and Sox-11 Involved in Autoinhibition of DNA Binding and Partnership Specificity

Sox transcription factors play key regulatory roles throughout development, binding DNA through a consensus (A/T)(A/T)CAA(A/T)G sequence. Although many different Sox proteins bind to this sequence, it has been observed that gene regulatory elements are commonly responsive to only a small subset of the entire family, implying that regulatory mechanisms exist to permit selective DNA binding and/or transactivation by Sox family members. To identify and explore the mechanisms modulating gene activation by Sox proteins further, we compared the function of Sox-2 and Sox-11. This led to the discovery that Sox proteins are regulated differentially at multiple levels, including transactivation, protein partnerships with Pit-Oct-Unc (POU) transcription factors, and DNA binding autoregulation. Specifically, we determined that Sox-11 activates transcription more strongly than Sox-2 and that the transactivation domain of Sox-11 is primarily responsible for this capability. Additionally, we demonstrate that the Sox-11 DNA binding domain is responsible for selective cooperation with the POU factor Brn-2. This requirement cannot be replaced by the DNA binding domain of Sox-2, indicating that the DNA binding domain of Sox proteins is critical for Sox-POU partnerships. Interestingly, we have also determined that a conserved domain of Sox-11 has the novel capability of autoinhibiting its ability to bind DNA in vitro and to activate gene expression in vivo. Our findings suggest that the autoinhibitory domain can repress promiscuous binding of Sox-11 to DNA and plays an important role in regulating the recruitment of Sox-11 to specific genes.

The nuclear protein HMGB1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway.

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.

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.

The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.