Nonhistone Chromosomal Proteins Research Articles

Abstract SY02-03: Chromatin architecture and gene regulation in oncogene-induced senescence.

Abstract SY02-03: Chromatin architecture and gene regulation in oncogene-induced senescence.

Interaction between DNA and chromosomal proteins HMGB1 and H1 studied by IR/VCD spectroscopy

Binary complexes of calf thymus DNA with calf thymus non-histone chromosomal protein HMGB1 and linker histone H1 were studied using FTIR/VCD spectroscopy. The spectroscopic data showed that the interaction of the protein HMGB1 and histone H1 with DNA resulted in formation of two different types of the macromolecular complexes. Histone H1 retained its native structure even at high concentrations and induced DNA condensation upon binding at the protein to DNA ratio r (w/w) in the complex r⩾0.3. HMGB1 demonstrated the ability to form soluble complexes at considerably higher protein to DNA ratios. The obtained data suggest that the HMGB1 also participated in the protein–protein interactions via its C-terminal domain.

Nucleosome structural changes induced by binding of non-histone chromosomal proteins HMGN1 and HMGN2

Interactions between the nucleosome and the non-histone chromosomal proteins (HMGN1 and HMGN2) were studied by circular dichroism (CD) spectroscopy to elucidate structural changes in the nucleosome induced by HMGN binding. Unlike previous studies that used a nucleosome extracted from living cells, in this study we utilized a nucleosome reconstituted from unmodified recombinant histones synthesized in Escherichia coli and a 189-bp synthetic DNA fragment harboring a nucleosome positioning sequence. This DNA fragment consists of 5′-TATAAACGCC-3′ repeats that has a high affinity to the histone octamer. A nucleosome containing a unique octamer-binding sequence at a specific location on the DNA was produced at sufficiently high yield for spectroscopic analysis. CD data have indicated that both HMGN1 and HMGN2 can increase the winding angle of the nucleosome DNA, but the extent of the structural changes induced by these proteins differs significantly. This suggests HMGN1 and HMGN2 would have different abilities to facilitate nucleosome remodeling.

HMGB1 and HMGB2 Expression Patterns in Human Papillary Renal Cancers

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 has a dual function, a cytokine in addition to a nuclear protein. The increased expression of HMGB1 has been reported for several different tumors. Here, we assessed HMGB1 and HMGB2 expressions in two cases of papillary renal cell carcinoma. One case with pT1a, Grade 2 showed HMGB1 expression in the nucleus and cytosol and HMGB2 expression in the nucleus, but not in the cytosol. In the other case, there were three renal tumors, one of which was clear cell renal cell carcinoma with pT1a, Grade 3 and two were papillary renal cell carcinomas, Grade 2 (5 mmand2 mmin the diameter). Both HMGB1 and HMGB2 were expressed in the nucleus and cytosol of papillary carcinoma. In the clear cell carcinoma of this case, HMGB1 expression was stained both in the nucleus and cytosol, while HMGB2 was observed in the nucleus, but not in the cytosol. More samples need to be further investigated in order to draw conclusions concerning HMGB expressions in papillary renal cell carcinomas.

Структура комплексов ДНК с хромосомным белком HMGB1 и гистоном Н1 в присутствии ионов марганца. II. Спектроскопия кругового дихроизма в ИК области

Complexes of DNA with nonhistone chromosomal protein HMGB1 and histone H1 in the presence of manganese ions were studied using absorption and circular dichroism spectroscopy in infrared region. It was shown that the approach provides good results for solutions containing large particles, which cause light scattering in UV region. It was also shown that the manganese ions are able to coordinate to the chemical groups of DNA as well as to the carboxylic amino acid residues of the protein HMGB1. The latter stimulates DNA condensation and slightly weakens DNA-protein interactions in the complex.

HMGB1 and HMGB2 Expression Patterns in Human Papillary Renal Cancers

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 has a dual function, a cytokine in addition to a nuclear protein. The increased expression of HMGB1 has been reported for several different tumors. Here, we assessed HMGB1 and HMGB2 expressions in two cases of papillary renal cell carcinoma. One case with pT1a, Grade 2 showed HMGB1 expression in the nucleus and cytosol and HMGB2 expression in the nucleus, but not in the cytosol. In the other case, there were three renal tumors, one of which was clear cell renal cell carcinoma with pT1a, Grade 3 and two were papillary renal cell carcinomas, Grade 2 (5 mmand2 mmin the diameter). Both HMGB1 and HMGB2 were expressed in the nucleus and cytosol of papillary carcinoma. In the clear cell carcinoma of this case, HMGB1 expression was stained both in the nucleus and cytosol, while HMGB2 was observed in the nucleus, but not in the cytosol. More samples need to be further investigated in order to draw conclusions concerning HMGB expressions in papillary renal cell carcinomas.

Dynamics of the HP1β-PCNA-containing complexes in DNA replication and repair

Heterochromatin protein 1 (HP1), a small non-histone chromosomal protein, was recently shown to form a complex in vivo with Proliferating Cell Nuclear Antigen (PCNA), a key factor in DNA replication. The complex, which requires HP1β in a form of a dimer, is engaged in DNA repair and replication. We now provide further evidence based on FRET-FLIM live cell studies confirming the association and close proximity between HP1β and PCNA in the complex. We also demonstrate using FRAP, that although HP1β-PCNA complexes are highly mobile in nonreplicating nuclei, when engaged in DNA replication, they become bound and do not exchange with the mobile pool. These observations are in agreement with a notion that a subpopulation of HP1 molecules interact with PCNA in vivo during DNA replication. Similarly, HP1β which is associated with PCNA in regions of DNA repair, is bound and does not exchange with the mobile pool, suggesting that HP1β in association with PCNA may be a component of a DNA repair complex.

A heterochromatin protein 1 (HP1) dimer and a proliferating cell nuclear antigen (PCNA) protein interact in vivo and are parts of a multiprotein complex involved in DNA replication and DNA repair

Heterochromatin protein 1 (HP1) is a small non-histone chromosomal protein known as a dominant suppressor of position-effect variegation and a major component of heterochromatin. Posttranslationally modified HP1, through interaction with protein partners from different groups, can be involved in a number of nuclear processes, including gene activation, chromatin remodeling, replication and DNA repair. Using bimolecular fluorescence complementation assay and live cell imaging, we demonstrate that HP1β and PCNA, a key player in DNA replication, are closely spaced components of a multiprotein complex involved in replication, both in S phase and during DNA repair, and that the functional complex requires formation of an HP1 dimer.

Anti-high mobility group box 1 and box 2 non-histone chromosomal proteins (HMGB1/HMGB2) antibodies and anti-Saccharomyces cerevisiae antibodies (ASCA): accuracy in differentially diagnosing UC and CD and correlation with inflammatory bowel disease phenotype

The development of a supportive diagnostic method has long been required to differentially diagnose ulcerative colitis (UC) and Crohn's disease (CD). Several antibodies circulate in the sera of patients with inflammatory bowel disease. We previously identified the high mobility group box 1 and box 2 non-histone chromosomal proteins (HMGB1 and HMGB2) as novel antigens of perinuclear type anti-neutrophil cytoplasmic antibodies (pANCA) and discovered anti-HMGB1/HMGB2 antibodies in sera from patients with UC. Here, we evaluated the ability of anti-HMGB1/HMGB2 antibodies combined with anti-Saccharomyces cerevisiae antibodies (ASCA) to differentially diagnose UC and CD. We measured titers of anti-HMGB1/HMGB2 antibodies and ASCA in the sera of 213 patients with UC and 93 with CD, using enzyme-linked immunosorbent assays. Among the patients with UC, 26.8% were positive for anti-HMGB1/HMGB2 antibodies, with 85.0% specificity towards CD and a positive predictive value of 80.3%. Corticosteroids significantly suppressed the titer of anti-HMGB1/HMGB2 antibodies. Among the patients with CD, 24.7% were positive for ASCA, with 96.2% specificity towards UC and a positive predictive value of 74.2%. Interestingly, the positivity rate of anti-HMGB/HMGB2 antibodies was higher (35.7%) in patients with the ileitis type of CD than in patients with CD in the colon (6.2%; significant difference, P<0.01). The specificity of anti-HMGB1/HMGB2 antibodies in UC for CD in the colon was 93.8%. CD in the colon and UC can be differentially diagnosed using anti-HMGB/HMGB2 antibodies combined with ASCA.

High mobility group in A1a (HMGA1a) small interfering RNA (siRNA) blockage decreases metastatic potential in cultured osteosarcoma cell lines

High mobility group A1 (HMGA1) is a non-histone chromosomal nuclear protein that plays important roles on gene transcription, recombination, and chromatin structure stabilization. The aim of this study was to investigate the effect of small interfering RNA (siRNA) on HMGA1 expression and metastatic potential in osteosarcoma cell in vivo. We obtained osteosarcoma cell sublines after single cell cloning technique from human osteosarcoma cell line MG-63, and chose the cell subline with high HMGA1 expression. One sequence-specific siRNA targeted to HMGA1 was designed and cloned to generate the pU6mRFP-HMGA1 shRNA vector, then transfected into the osteosarcoma cell subline. The transfection efficiency of the experiment group and control group were 55.68±6.74 and 49.87±4.33%. The specific HMGA1 shRNA effectively down regulated the mRNA and the protein expression of osteosarcoma cell subline. The cells numbers throwing membrane at the experiment group was higher than that of the control group. shRNA of HMGA1 could successfully inhibit the expression of target gene in mRNA and protein levels and inhibit the in vitro metastatic potential of osteosarcoma cells. Key words: RNA interference, osteosarcoma cell, high mobility group A.

Expression of High Mobility GroupA2 is Associated with Poor Survival in Hepatocellular Carcinoma

Primary hepatocellular carcinoma (HCC) is one of the most common solid malignancies [1]. Despite recent improvements in surveillance, diagnosis and clinical treatment strategies, HCC prognosis remains dismal. Patients often experience high metastasis or recurrence rates, and postoperative 5-year survival ranges from 17 % to 53 % [2]. To date, many studies have reported aberrant gene expression in HCC, but molecular factors identified as potential therapeutic targets remain limited. Therefore, it is a challenging task to identify relevant biomarkers or potential pharmacological targets in clinical practice. The high mobility group A2 (HMGA2; also called HMGI-C) protein, a member of the high mobility group AT-hook (HMGA) family, is a small non-histone chromosomal protein that has no intrinsic transcriptional activity, but can modulate transcription by binding to DNA and altering chromatin architecture [3–5]. By interacting with many different transcription factors, HMGA2 is an important regulator of cell growth, differentiation, apoptosis, and transformation [5]. HMGA2 protein is expressed at a high level during embryogenesis, but is hardly detectable in adult human tissues [6, 7]. HMGA2 is frequently involved in chromosomal translocations that occur in benign human tumors, such as lipomas [8], uterine leiomyomas [9], and lung hamartomas [10]. Furthermore, increased HMGA2 expression has been detected in various human epithelialtype tumors, including breast cancers [11], lung cancers [12, 13], pancreatic carcinomas [14], thyroid carcinomas [15], and gastric cancers [16]. These studies suggest that HMGA2 protein overexpression may lead to neoplastic transformation. However, there have been no reports of HMGA2 expression in hepatocellular carcinoma. In this study, we investigated HMGA2 expression in HCC to determine its clinicopathologic and prognostic value.

Microglia in Alzheimer's Disease

Microglia in Alzheimer's Disease

Interaction between Nonhistone Protein HMGB1 and Linker Histone H1 Facilitates the Formation of Structurally Ordered DNA‐Protein Complexes

The structural organization of the DNA complexes with nonhistone chromosomal protein and linker histone H1 was studied using circular dichroism spectroscopy (CD) and atomic force microscopy (AFM). It has been shown that due to the interaction between HMGB1 and H1 highly ordered DNA‐protein complexes emerge in the solution. Their spectral properties are found to be similar to those of DNA/HMGB1‐(AB) complexes, reported earlier. AFM images reveal the formation of fibril‐like structures in the solution. We suggest that the electrostatic screening of the HMGB1 C‐terminal domain by histone H1 facilitates stronger interaction of the HMGB1/H1 with DNA and the formation of the ordered supramolecular DNA‐protein complexes.

Molecular characterization of heterochromatin proteins 1a and 1b from the silkworm, Bombyx mori

Heterochromatin protein 1s (HP1s) are nonhistone chromosomal proteins that play a direct role in the formation and maintenance of heterochromatin structure. Similarly to Caenorhabditis elegans, silkworms possess holocentric chromosomes, in which diffused kinetochores extend along the length of each chromosome. We have isolated two silkworm HP1 homologues, BmHP1a and BmHP1b. Cytological analysis showed a unique localization of BmHP1s during cell division, in which these proteins first appear to dissociate from the chromosomes, but then return to enclose the chromosomes during metaphase. BmHP1s formed homo- and hetero-dimers and interacted with BmSu(var)3-9, which is a methyltransferase for histone H3 lysine 9 (H3K9). We further showed, using a silkworm cell-based reporter system, that BmHP1b had higher transcriptional repression activity than BmHP1a, whereas BmHP1a interacted more strongly with BmSu(var)3-9 than did BmHP1b. These results suggest that silkworm HP1a and HP1b may play different roles in heterochromatin formation in holocentric silkworm chromosomes.

Comprehensive analysis of AHL homologous genes encoding AT-hook motif nuclear localized protein in rice

The AT-hook motif is a small DNA-binding protein motif that has been found in the high mobility group of non-histone chromosomal proteins. The Arabidopsis genome contains 29 genes encoding the AT-hook motif DNA-binding protein (AHL). Recent studies of Arabidopsis genes (AtAHLs) have revealed that they might play diverse functional roles during plant growth and development. In this report, we mined 20 AHL genes (OsAHLs) from the rice genome database using AtAHL genes as queries and characterized their molecular features. A phylogenetic tree revealed that OsAHL proteins can be classified into 2 evolutionary clades. Tissue expression pattern analysis revealed that all of the OsAHL genes might be functionally expressed genes with 3 distinct expression patterns. Nuclear localization analysis using transgenic Arabidopsis showed that several OsAHL proteins are exclusively localized in the nucleus, indicating that they may act as architectural transcription factors to regulate expression of their target genes during plant growth and development.

MP-03.03 HMGA2 Upregulation Is Related To Tumor Progression and let-7 Downregulation in High Risk Prostate Carcinoma

The highmobility group A2 (HMGA2) nonhistone chromosomal protein can modulate transcription by altering chromatin architecture. HMGA2 is highly expressed during embryogenesis and in various benign and malignant tumors. Recent studies report that HMGA2 is negatively regulated by the let-7 microRNA (miRNA) family. However, no studies have examined the clinical significance of HMGA2 expression and its relationship to the let-7 miRNA family in prostate cancer.

Role of High Mobility Group Box 1 (HMGB1) in Wound Healing

HMGB1, a non-histone chromosomal protein, can bind to the receptor for advanced glycation end products (RAGE) and act as an inflammatory mediator. We examined the role of HMGB1 in incisional wound healing and its possible mechanism of action through receptor for advanced glycation end products (RAGE). Male Sprague-Dawley rats undergoing full-thickness incisional wounding with subcutaneous implantation of PVA sponges were given daily injections of ethyl pyruvate (EP) (40 mg/kg, i.p.), a potent inhibitor of HMGB1 release. At 7 d post-wounding, wound breaking strength, sponge collagen content, and wound fluid HMGB1 levels were assessed. In vitro rat dermal or wound-derived fibroblasts were cultured with recombinant HMGB1 or advanced glycation end product (AGE). Some cultures were co-treated with a RAGE-blocking antibody. Fibroblast proliferation and collagen synthesis were assayed. In vivo treatment with EP significantly decreased wound HMGB1 levels (P < 0.05), which was paralleled by increased wound breaking strength (P < 0.05) and wound collagen content (P < 0.05). In vitro treatment with HMGB1 (100 ng/mL) had no effect on fibroblast proliferation but significantly reduced collagen synthesis (P < 0.05). This effect was abrogated by co-treatment with anti-RAGE antibody. Fibroblasts treated with AGE had lower collagen synthesis (P < 0.01), which was restored by anti-RAGE antibody treatment. HMGB1 impairs fibroblast collagen synthesis. Reducing wound HMGB1 levels lead to increased tensile strength and collagen synthesis. The data suggest that HMGB1 affects collagen synthesis through activation of RAGE.

Abstract 206: High mobility group A1(HMGA1) proteins localize at the mitochondria and inhibit the p53-mediated intrinsic-apoptotic pathway

Abstract The high mobility group A (HMGA) non-histone chromosomal proteins play key roles in chromatin architecture and orchestrate the assembly of nucleoprotein complexes involved in gene transcription, replication, and chromatin structure. HMGA overexpression and gene rearrangements are frequent events in human cancer, but the molecular basis of HMGA oncogenic activity has not been completely elucidated. Recently, we have defined a new physical and functional interaction between HMGA1 and p53. This interaction modulates the transcription of p53 target genes such as Mdm2, p21waf1, Bax, Bcl-2, and inhibits p53-mediated apoptosis. Here we report that HMGA1 does not act only at transcriptional level but it has also a direct, anti-apoptogenic role at the mitochondria. In fact, we found that in mouse embryonic fibroblast (MEF) and several tumour cell lines overexpressing HMGA1, this protein is present at cytoplasmic level other than in the nucleus. Moreover, we demonstrate that HMGA1 stably localizes at the mitochondria, in which it binds Bcl-2, displacing it from the binding to p53, thus counteracting the release of cytochrome C. In agreement with this observation, the HMGA1-overexpressing cells show a significant reduction of cytochrome C release, after lethal dose of Ultraviolet (UV) light, whereas HMGA1-depleted cells are more susceptibile to UV treatment. These data define a new localization of HMGA1 at the mitochondria level that allows HMGA1 to inhibit by a new mechanism the p53-mediated intrinsic-apoptotic pathway. This mechanism may also have a role in the resistance of malignant cells to chemo- and radio-therapy, in particularly for the tumor cells harboring wild-type form of p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2011-206

The HP1a Disordered C Terminus and Chromo Shadow Domain Cooperate to Select Target Peptide Partners

Drosophila melanogaster heterochromatin protein 1a (HP1a) is essential for compacted heterochromatin structure and the associated gene silencing. Its chromo shadow domain (CSD) is well known for binding to peptides that contain a PXVXL motif. Heterochromatin protein 2 (HP2) is a non-histone chromosomal protein that associates with HP1a in the pericentric heterochromatin, telomeres, and the fourth chromosome. Using NMR spectroscopy, fluorescence polarization, and site-directed mutagenesis, we identified an LCVKI motif in HP2 that binds to the HP1a CSD. The binding affinity of the HP2 fragment is approximately two orders of magnitude higher than that of peptides from PIWI (with a PRVKV motif), AF10 (with a PLVVL motif), or CG15356 (with LYPLL and LSIVA motifs). To delineate differential interactions of the HP1a CSD, we characterized its structure, backbone dynamics, and dimerization constant. We found that the dimerization constant is bracketed by the affinities of HP2 and PIWI, which dock to the same HP1a homodimer surface. This suggests that HP2, but not PIWI, interaction can drive the homodimerization of HP1a. Interestingly, the integrity of the disordered C-terminal extension (CTE) of HP1a is essential for discriminatory binding, whereas swapping the PXVXL motifs does not confer specificity. Serine phosphorylation at the peptide binding surface of the CSD is thought to regulate heterochromatin assembly. Glutamic acid substitution at these sites destabilizes HP1a dimers, but improves the interaction with both binding partners. Our studies underscore the importance of CSD dimerization and cooperation with the CTE in forming distinct complexes of HP1a.

Conformational properties of nuclear protein HMGB1 and specificity of its interaction with DNA

Changes in the secondary structure of DNA and non-histone chromosomal protein HMGB1 were studied by circular dichroism and UV spectroscopy. We have demonstrated that the HMGBI protein is able to change its secondary structure upon binding to DNA. We estimated the proportion of bound protein on the assumption that there were two spectrally distinguishable forms of the HMGB1 in solution. The bound protein fraction decreases with increasing protein to DNA ratios (r) from 0.48 at r = 0.13 to 0.06 at r = 2.43. It has been shown that HMGB1 is able to induce considerable changes in DNA structure even when the amount of the protein directly associated with DNA is low.