High mobility group box 1 (HMGB1) protein plays multiple roles in transcription, replication, and cellular differentiation. HMGB1 is also secreted by activated monocytes and macrophages and passively released by necrotic or damaged cells, stimulating inflammation. HMGB1 is a novel antigen of antineutrophil cytoplasmic antibodies (ANCA) observed in the sera of patients with ulcerative colitis and autoimmune hepatitis, suggesting that HMGB1 is secreted from neutrophils to the extracellular milieu. However, the actual distribution of HMGB1 in the cytoplasm of neutrophils and the mechanisms responsible for it are obscure. Here we show that HMGB1 in neutrophils is post-translationally mono-methylated at Lys 42 . The methylation alters the conformation of HMGB1 and weakens its DNA binding activity, causing it to become largely distributed in the cytoplasm by passive diffusion out of the nucleus. Thus, post-translational methylation of HMGB1 causes its cytoplasmic localization in neutrophils. This novel pathway explains the distribution of nuclear HMGB1 to the cytoplasm and is important for understanding how neutrophils release HMGB1 to the extracellular milieu. High mobility group box 1 (HMGB1)2 protein is one of the most abundant nonhistone chromosomal proteins in eukaryotic organisms. The primary sequences of HMGB1 in various higher organisms, from birds to mammals, show more than 90% homology with each other (1). The protein has multiple roles in transcription, replication, and cellular differentiation (2, 3). HMGB1 interacts with several transcription factors, thereby allowing them to perform their cellular roles. The phenotype of Hmgb1 knock-out mice confirmed the functional importance of HMGB1 as a regulator of transcription: they die shortly after birth and show a defect in the transcriptional control exerted by the glucocorticoid receptor (4). The subcellular distribution of the protein is tissue-specific: HMGB1 is located in both the nuclei and the cytoplasm of different tissues, such as lymphoid tissue, testis, neurons, and hepatocytes (5). Wang et al. (6) identified HMGB1 as a late mediator of endotoxin lethality in mice and showed that monocytes and macrophages stimulated by lipopolysaccharide (LPS), tumor necrosis factor (TNF) or interleukin-1 (IL-1) secrete HMGB1 in a delayed response. Patients with sepsis show an increased serum level of HMGB1, which is correlated with the severity of infection (7). Moreover, HMGB1 in monocytes and macrophages is extensively acetylated upon activation by LPS, causing localization of the protein to the cytosol (8). Cytosolic HMGB1 is then concentrated into secretory lysosomes and secreted when the cells receive an appropriate second signal (9). The recent discovery of extracellular HMGB1 as a proinflammatory mediator has been supported by a number of studies. In addition, HMGB1 is passively released from the nucleus to the extracellular milieu by cells that die as a result of necrosis or damage (10).Our previous studies showed that HMGB1 and HMGB2 are novel antigens of a...

Gibberellins (GAs) are essential regulators of plant development, and DELLAs are negative regulators of GA signaling. The mechanism of GA-dependent transcription has been explained by DELLA-mediated titration of transcriptional activators and their release through the degradation of DELLAs in response to GA. However, the effect of GA on genome-wide expression is predominantly repression, suggesting the existence of unknown mechanisms of GA function. In this study, we identified an Arabidopsis thaliana DELLA binding transcription factor, GAI-ASSOCIATED FACTOR1 (GAF1). GAF1 shows high homology to INDETERMINATE DOMAIN1 (IDD1)/ENHYDROUS. GA responsiveness was decreased in the double mutant gaf1 idd1, whereas it was enhanced in a GAF1 overexpressor. GAF1 binds to genes that are subject to GA feedback regulation. Furthermore, we found that GAF1 interacts with the corepressor TOPLESS RELATED (TPR) and that DELLAs and TPR act as coactivators and a corepressor of GAF1, respectively. GA converts the GAF1 complex from transcriptional activator to repressor via the degradation of DELLAs. These results indicate that DELLAs turn on or off two sets of GA-regulated genes via dual functions, namely titration and coactivation, providing a mechanism for the integrative regulation of plant growth and GA homeostasis.

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HMGB1, a nonhistone chromosomal protein in higher eukaryotic nuclei, consists of two DNA binding motifs called HMG boxes and an acidic C-tail comprising a continuous array of 30 acidic amino acid residues. In the preceding study, we showed that the acidic C-tail of HMGB1 is required for transcription stimulation accompanied by chromatin decondensation in cultured cells. However, details of the involvement of the acidic C-tail in transcription stimulation were not clear. To clarify the mechanism of transcription stimulation by the acidic C-tail, we assessed the effect of the acidic C-tail on the transcription stimulation and nucleosome binding. Transcription stimulation assays using acidic C-tail deletion mutants showed that the five amino acid residues at the C-terminal end of HMGB1, a DDDDE sequence, are essential for the stimulation. The DDDDE sequence was also required for the preferential binding of HMGB1 to nucleosome linker DNA, which is a cognate HMGB1 binding site in chromatin. Cross-linking and far-Western experiments demonstrated that the DDDDE sequence interacts with the core histone H3 N-tail. These results strongly suggest that the interaction between the DDDDE sequence of HMGB1 and the H3 N-tail is a key factor for the transcription stimulation by HMGB1 as well as the preferential binding of HMGB1 to chromatin.

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.

Several in vitro studies have suggested that high mobility group (HMG) protein 1 has a role in gene regulation as a trans activator or quasi-transcription factor. However, data on the molecular functions of HMG1 protein in these reactions are contradictory or obscure. In order to assess whether HMG1 protein does, in fact, have transcriptional activation potential, two assay systems in cultured cells were employed. HMG1 protein introduced into COS-1 cells as a complex with a reporter plasmid carrying the lacZ gene enhanced the level of the gene expression. Cotransfection of an expression plasmid carrying HMG1 cDNA into the cells with the reporter plasmid enhanced the activity of beta-galactosidase 2-3-fold in comparison with that of the control effector plasmid. The enhancement was proved to be dependent not on the replication but on the transcription of the reporter plasmid. In the cotransfection experiments, an expression plasmid the HMG1 molecule lacking the acidic carboxyl terminus repressed the expression of the reporter gene. The binding of an HMG1 protein variant lacking the acidic carboxyl terminus to DNA gave an extremely large shift of gel retardation in comparison with the complete HMG1 molecule. Together, these results indicate that HMG1 protein can enhance expression in cells in culture at the step of gene transcription and that the DNA binding domains comprising two-thirds of the HMG1 protein molecule are responsible for the inhibition property. Also, the acidic terminus of the HMG1 molecule is essential for the enhancement of gene expression in addition to elimination of the repression caused by the DNA binding. (ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARYAnti-neutrophil cytoplasmic antibodies (ANCA) in sera from ulcerative colitis (UC) patients have been described as reacting with proteins in the granules of human neutrophils such as cathepsin G and lactoferrin and with yet unidentified antigens. Here we report the existence of a new member of perinuclear ANCA (P-ANCA) in UC patients. In the previous study, we found that UC patients had a novel P-ANCA against neutrophil 28-kD protein. In this study, we purified the same antigens from HL-60 lysates by using reversed phase high-performance liquid chromatography, and revealed that the 28-kD antigen consisted of two different proteins. The N-terminus amino acids of these proteins are identical with those of high mobility group (HMG) non-histone chromosomal proteins HMG1 and HMG2. Immunoblotting analysis of human neutrophil lysates using rabbit anti-HMG1/2 antisera revealed a single band of 28 kD, and the 28-kD band detected by immunoblotting analysis using patient's serum IgG completely disappeared after preincubation with a mixture of HMG1 and HMG2. Furthermore, rabbit anti-HMG1/2 antisera showed a perinuclear staining pattern in indirect immunofluorescence studies using ethanol-fixed neutrophils. These data demonstrate that HMG1 and HMG2 are novel target antigens of P-ANCA. HMG1 and HMG2 are distributed in the nuclei and cytoplasm of eukaryotic cells and act as transcription factors. Their intracellular localization and functions are distinct from those of the previously reported granular antigens of P-ANCA.

We developed murine C-127 cell lines that stationarily overexpress high mobility group (HMG) proteins 1 and 2 by transfecting them with the bovine papilloma virus plasmid carrying their respective cDNA sequences. Using these cell lines, we examined the effects of these HMG proteins on the modulation of chromatin structure that accompanied transcription. The levels of HMG1 mRNA and protein in cells overexpressing HMG1 protein were enhanced about 7- and 3-fold, respectively, in comparison with control cells, whereas those in cells overexpressing HMG2 protein were enhanced about 17- and 9-fold. The expression of reporter genes transfected into the cells was enhanced approximately 2-fold in cells overexpressing HMG1, but not HMG2, in comparison with those in control cells, irrespective of the sources of the genes and promoters. The minichromosome derived from the reporter plasmid in cells overexpressing HMG1 protein was more susceptible to micrococcal nuclease digestion than those in cells overexpressing HMG2 protein and control cells. The enhanced accessibility to micrococcal nuclease was not restricted to the expressing gene and promoter but involved the entire minichromosome, suggesting that the enhancement of gene expression resulted from changes in the condensation of the entire minichromosomal region by HMG1 protein. Minichromosomes in cells overexpressing HMG contained enhanced amounts of the respective HMG proteins and simultaneously reduced amounts of histone H1s. These results suggest that HMG1 and -2 proteins have different functions in the modulation of chromatin structure, and that HMG1 protein may sustain the structure of the respective gene to ensure that its activity as a template is expressed fully. These observations on the modulation of chromatin structure accompanying gene transcription in cells overexpressing HMG protein may provide important information on the function of these proteins.