The Effect of HMGB1 and HMGB2 on Transcriptional Regulation Differs in Neuroendocrine and Adenocarcinoma Models of Prostate Cancer.

Human high-mobility group-B (HMGB) proteins regulate gene expression in prostate cancer (PCa), a leading cause of oncological death in men. Their role in aggressive PCa cancers, which do not respond to hormonal treatment, was analyzed. The effects of HMGB1 and HMGB2 silencing upon the expression of genes previously related to PCa were studied in the PCa cell line PC-3 (selected as a small cell neuroendocrine carcinoma, SCNC, PCa model not responding to hormonal treatment). A total of 72% of genes analyzed, using pre-designed primer panels, were affected. HMGB1 behaved mostly as a repressor, but HMGB2 as an activator. Changes in SERPINE1, CDK1, ZWINT, and FN1 expression were validated using qRT-PCR after HMGB1 silencing or overexpression in PC-3 and LNCaP (selected as an adenocarcinoma model of PCa responding to hormonal treatment) cell lines. Similarly, the regulatory role of HMGB2 upon SERPINE1, ZWINT, FN1, IGFPB3, and TYMS expression was validated, finding differences between cell lines. The correlation between the expression of HMGB1, HMGB2, and their targets was analyzed in PCa patient samples and also in PCa subgroups, classified as neuroendocrine positive or negative, in public databases. These results allow a better understanding of the role of HMGB proteins in PCa and contribute to find specific biomarkers for aggressive PCa.

Thanksgiving to Yeast, the HMGB Proteins History from Yeast to Cancer.

Yeasts have been a part of human life since ancient times in the fermentation of many natural products used for food. In addition, in the 20th century, they became powerful tools to elucidate the functions of eukaryotic cells as soon as the techniques of molecular biology developed. Our molecular understandings of metabolism, cellular transport, DNA repair, gene expression and regulation, and the cell division cycle have all been obtained through biochemistry and genetic analysis using different yeasts. In this review, we summarize the role that yeasts have had in biological discoveries, the use of yeasts as biological tools, as well as past and on-going research projects on HMGB proteins along the way from yeast to cancer.

Translocated HMGB3 is involved in papillary thyroid cancer progression by activating cytoplasmic TLR3 and transmembrane TREM1.

The family of high mobility group box (HMGB) proteins participates in various biological processes including immunity, inflammation, as well as cancer formation and progression. However, its role in thyroid cancer remains to be clarified. We performed quantitative RT-PCR (qRT-PCR), western blot, enzyme-linked immunosorbent, immunohistochemistry, and immunofluorescence assays to evaluate the expression level and subcellular location of HMGB3. The effects of HMGB3 knockdown on malignant biological behaviors of thyroid cancer were determined by cell proliferation assays, cell cycle and apoptosis assays, and transwell chamber migration and invasion assays. Differential expression genes (DEGs) altered by HMGB3 were analyzed using the Ingenuity Pathway Analysis (IPA) and TRRUST v2 database. HMGB3 correlated pathways predicted by bioinformatic analysis were then confirmed using western blot, co-immunoprecipitation, dual-luciferase reporter assay, and flow cytometry. We found that HMGB3 is overexpressed and its downregulation inhibits cell viability, promotes cell apoptosis and cell cycle arrest, and suppresses cell migration and invasion in thyroid cancer. In PTC, both tissue and serum levels of HMGB3 are elevated and are correlated with lymph node metastasis and advanced tumor stage. Mechanistically, we observed the translocation of HMGB3 in PTC, induced at least partially by hypoxia. Cytoplasmic HMGB3 activates nucleic-acid-mediated TLR3/NF-κB signaling and extracellular HMGB3 interacts with the transmembrane TREM1 receptor in PTC. This study demonstrates the oncogenic role of HMGB3 cytoplasmic and extracellular translocation in papillary thyroid cancers; we recommend its future use as a potential circulating biomarker and therapeutic target for PTC.

A viral histone-like protein exploits antagonism between linker histones and HMGB proteins to obstruct the cell cycle.

Virus infection necessarily requires redirecting cellular resources toward viral progeny production. Adenovirus encodes the histone-like protein VII, which causes catastrophic global reorganization of host chromatin to promote virus infection. Protein VII recruits the family of high mobility group box (HMGB) proteins to chromatin along with the histone chaperone SET. As a consequence of this recruitment, we find that protein VII causes chromatin depletion of several linker histone H1 isoforms. The relationship between linker histone H1 and the functionally opposite HMGB proteins is critical for higher-order chromatin structure. However, the physiological consequences of perturbing this relationship are largely unknown. Here, we employ complementary systems in Saccharomyces cerevisiae and human cells to demonstrate that adenovirus protein VII disrupts the H1-HMGB balance to obstruct the cell cycle. We find that protein VII causes an accumulation of G2/M cells both in yeast and human systems, underscoring the high conservation of this chromatin vulnerability. In contrast, adenovirus E1A and E1B proteins are well established to override cell cycle regulation and promote transformation of human cells. Strikingly, we find that protein VII obstructs the cell cycle, even in the presence of E1A and E1B. We further show that, in a protein-VII-deleted infection, several cell cycle markers are regulated differently compared to wild-type infection, supporting our model that protein VII plays an integral role in hijacking cell cycle regulation during infection. Together, our results demonstrate that protein VII targets H1-HMGB1 antagonism to obstruct cell cycle progression, revealing an unexpected chromatin vulnerability exploited for viral benefit.

Differential Characteristics of HMGB2 Versus HMGB1 and their Perspectives in Ovary and Prostate Cancer.

We have summarized common and differential functions of HMGB1 and HMGB2 proteins with reference to pathological processes, with a special focus on cancer. Currently, several "omic" approaches help us compare the relative expression of these 2 proteins in healthy and cancerous human specimens, as well as in a wide range of cancer-derived cell lines, or in fetal versus adult cells. Molecules that interfere with HMGB1 functions, though through different mechanisms, have been extensively tested as therapeutic agents in animal models in recent years, and their effects are summarized. The review concludes with a discussion on the perspectives of HMGB molecules as targets in prostate and ovarian cancers.

Association between high-mobility group box B1 and hepatic ischemia-reperfusion injury / 临床肝胆病杂志

High-mobility group box B1 (HMGB1) is a DNA-binding protein widely distributed in eukaryotic cells. When tissue damage occurs, HMGB1 acts as an endogenous risk signal to activate the body’s immune system and mediate aseptic inflammatory response. Current research findings have shown that HMGB1 plays a key role in hepatic ischemia-reperfusion injury. This article summarizes the recent research advances in the proinflammatory role of HMGB1 in hepatic ischemia-reperfusion and HMGB1 as a target for the treatment of hepatic ischemia-reperfusion injury.

Association between high-mobility group box B1 and hepatic ischemia-reperfusion injury / 临床肝胆病杂志

High-mobility group box B1 (HMGB1) is a DNA-binding protein widely distributed in eukaryotic cells. When tissue damage occurs, HMGB1 acts as an endogenous risk signal to activate the body’s immune system and mediate aseptic inflammatory response. Current research findings have shown that HMGB1 plays a key role in hepatic ischemia-reperfusion injury. This article summarizes the recent research advances in the proinflammatory role of HMGB1 in hepatic ischemia-reperfusion and HMGB1 as a target for the treatment of hepatic ischemia-reperfusion injury.

[HMGB Proteins as DNA Chaperones That Modulate Chromatin Activity].

HMGB proteins are involved in structural rearrangements caused by regulatory chromatin remodeling factors. Particular interest is attracted to a DNA chaperone mechanism, suggesting that the HMGB proteins introduce bends into the double helix, thus rendering DNA accessible to effector proteins and facilitating their activity. The review discusses the role that the HMBG proteins play in key intranuclear processes, including assembly of the preinitiation complex during transcription of ribosomal genes; transcription by RNA polymerases I, II, and III; recruitment of the SWI/SNF complex during transcription of nonribosomal genes; DNA repair; etc. The functions of the HMGB proteins are considered in detail with the examples of yeast HMO1 and NHP6. The two proteins possess unique features in adition to properties characteristic of the HMGB proteins. Thus, NHP6 stimulates a large-scale ATP-independent unwrapping of nucleosomal DNA by the FACT complex, while in its absence FACT stabilizes the nucleosome. HMO1 acts as an alternative linker histone. Both HMO1 and NHP6 are of applied interest primarly because they are homologs of human HMGB1, an important therapeutic target of anticancer and anti-inflammatory treatments.

Elevated Serum High-Mobility Group Box-1 Protein Level Is Associated with Poor Functional Outcome in Ischemic Stroke.

BACKGROUND: In experimental models, inhibition of high-mobility group box-1 (HMGB1) signaling has been reported to protect against the sequelae of ischemic stroke. Here, we determined the clinical significance of serum HMGB1 levels in patients with acute ischemic stroke. METHODS: We enrolled 183 patients (114 men, 69 women; mean age: 72.7 years) over 6 consecutive months. On admission and day 7, we recorded the National Institutes of Health Stroke Scale scores and measured serum high-sensitivity C-reactive protein (hs-CRP) and HMGB1 levels. Stroke volumes were estimated using diffusion-weighted magnetic resonance imaging performed on admission. One year later, clinical outcome was assessed using the modified Rankin Scale (mRS). RESULTS: Serum hs-CRP and HMGB1 levels in patients with ischemic stroke were increased relative to healthy controls (both P < .01). On day 7, hs-CRP, but not HMBG1, levels had increased significantly relative to levels at admission (P < .01 and .54, respectively). Higher HMGB1, but not hs-CRP, levels at day 7 correlated with larger stroke volumes (P < .01 and .28, respectively). HMGB1 levels did not significantly differ between stroke subtypes. Multiple logistic regression analysis indicated that a serum HMGB1 level higher than 7.5 ng/mL was an independent risk factor for poor prognosis, defined as a 1-year mRS score of 3-6 (odds ratio, 2.34; 95% confidence interval, 1.02-5.38). CONCLUSIONS: Acute ischemic stroke is associated with elevated serum HMGB1 levels, and HMGB1 levels at admission independently predict poor outcome at 1 year. These results suggest that HMGB1 quantification provides more accurate prognostic information after ischemic stroke.

Yeast HMO1: Linker Histone Reinvented.

Eukaryotic genomes are packaged in chromatin. The higher-order organization of nucleosome core particles is controlled by the association of the intervening linker DNA with either the linker histone H1 or high mobility group box (HMGB) proteins. While H1 is thought to stabilize the nucleosome by preventing DNA unwrapping, the DNA bending imposed by HMGB may propagate to the nucleosome to destabilize chromatin. For metazoan H1, chromatin compaction requires its lysine-rich C-terminal domain, a domain that is buried between globular domains in the previously characterized yeast Saccharomyces cerevisiae linker histone Hho1p. Here, we discuss the functions of S. cerevisiae HMO1, an HMGB family protein unique in containing a terminal lysine-rich domain and in stabilizing genomic DNA. On ribosomal DNA (rDNA) and genes encoding ribosomal proteins, HMO1 appears to exert its role primarily by stabilizing nucleosome-free regions or "fragile" nucleosomes. During replication, HMO1 likewise appears to ensure low nucleosome density at DNA junctions associated with the DNA damage response or the need for topoisomerases to resolve catenanes. Notably, HMO1 shares with the mammalian linker histone H1 the ability to stabilize chromatin, as evidenced by the absence of HMO1 creating a more dynamic chromatin environment that is more sensitive to nuclease digestion and in which chromatin-remodeling events associated with DNA double-strand break repair occur faster; such chromatin stabilization requires the lysine-rich extension of HMO1. Thus, HMO1 appears to have evolved a unique linker histone-like function involving the ability to stabilize both conventional nucleosome arrays as well as DNA regions characterized by low nucleosome density or the presence of noncanonical nucleosomes.

Dual function of Ixr1 in transcriptional regulation and recognition of cisplatin-DNA adducts is caused by differential binding through its two HMG-boxes.

Ixr1 is a transcriptional factor involved in the response to hypoxia, which is also related to DNA repair. It binds to DNA through its two in-tandem high mobility group box (HMG-box) domains. Each function depends on recognition of different DNA structures, B-form DNA at specific consensus sequences for transcriptional regulation, or distorted DNA, like cisplatin-DNA adducts, for DNA repair. However, the contribution of the HMG-box domains in the Ixr1 protein to the formation of different protein-DNA complexes is poorly understood. We have biophysically and biochemically characterized these interactions with specific DNA sequences from the promoters regulated by Ixr1, or with cisplatin-DNA adducts. Both HMG-boxes are necessary for transcriptional regulation, and they are not functionally interchangeable. The in-tandem arrangement of their HMG-boxes is necessary for functional folding and causes sequential cooperative binding to specific DNA sequences, with HMG-box A showing a higher contribution to DNA binding and bending than the HMG-box B. Binding of Ixr1 HMG boxes to specific DNA sequences is entropy driven, whereas binding to platinated DNA is enthalpy driven for HMG-box A and entropy driven for HMG-box B. This is the first proof that HMG-box binding to different DNA structures is associated with predictable thermodynamic differences. Based on our study, we present a model to explain the dual function of Ixr1 in the regulation of gene expression and recognition of distorted DNA structures caused by cisplatin treatment.

Serum levels of HMGB1 have a diagnostic role in metastatic renal cell cancer.

RCC constitutes approximately 90% of all renal malignancies and 2-3% of all malignant tumours in adults. In spite of the improvement in radiologic methods, nearly 30% of the early metastatic RCC patients are incidentally diagnosed. HMGB1 is an extracellular signalling molecule that plays a role both in inflammation and carcinogenesis. Patients who were followed in Medical Oncology Departments of Denizli Government Hospital and Antalya Education and Research Hospital with a histopathological diagnosis of RCC between years 2010-2012 were enrolled in this study. HMGB1 levels were also assessed in a manually performed quantitative sandwich-enzyme-linked immunosorbent assay (ELISA) assay kit. In our study, we showed that the serum level of HMGB1, whether 149.9 pg/ml or not is important in differential diagnosis between patient and control group.

Quantitative Evaluation of BAFF, HMGB1, TLR 4 AND TLR 7 Expression in Patients with Relapsing Remitting Multiple Sclerosis.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by a complex immune response. Because of the complex nature of MS pathogenesis, a panel of biomarkers derived from different platforms will be required to reflect disease-related alterations. Monitoring and evaluation of molecules associated with the pathogenesis of the disease would provide useful information on disease progression and therapeutic assessment. In view of this, we evaluated the mRNA expression levels of B-cell activating factor (BAFF), high mobility group box 1 (HMGB-1), Toll like receptor (TLR) 4 and TLR7 in MS. These molecules are implicated in the pathogenesis of MS; however, they havereceived little attention. PBMCs were isolated from whole blood of 84 Relapsing Remitting Multiple Sclerosis patients and 70 healthy controls. Relative quantitative RT-PCR was applied to quantify the transcriptional levels of the immune markers. The mRNA expression levels of TLR7 were significantly elevated in RRMS patients than healthy controls. Whereas, TLR4 expression was found to be significantly lower in the patients than control group. We found no difference analyzing the mRNA levels of BAFF and HMGB1. Our data highlights the immune marker correlates in RRMS patients. However, further in-depth studies are warranted to check for their reliability of biomarkers in autoimmune diseases such as MS.

The distinct C-terminal acidic domains of HMGB proteins are functionally relevant in Schistosoma mansoni.

The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins.

Nucleosome remodeling by the SWI/SNF complex is enhanced by yeast high mobility group box (HMGB) proteins.

The regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key factors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues.