HMGB Proteins Research Articles

The Non-Histone Protein FgNhp6 Is Involved in the Regulation of the Development, DON Biosynthesis, and Virulence of Fusarium graminearum.

Fusarium graminearum is the primary causative agent of Fusarium head blight (FHB), a devastating disease affecting cereals globally. The high-mobility group (HMG) of non-histone proteins constitutes vital architectural elements within chromatin, playing diverse roles in various biological processes in eukaryotic cells. Nonetheless, the specific functions of HMG proteins in F. graminearum have yet to be elucidated. Here, we identified 10 HMG proteins in F. graminearum and extensively characterized the biological roles of one HMGB protein, FgNhp6. We constructed the FgNhp6 deletion mutant and its complementary strains. With these strains, we confirmed the nuclear localization of FgNhp6 and discovered that the absence of FgNhp6 led to reduced radial growth accompanied by severe pigmentation defects, a significant reduction in conidial production, and a failure to produce perithecia. The ∆FgNhp6 mutant exhibited a markedly reduced pathogenicity on wheat coleoptiles and spikes, coupled with a significant increase in deoxynivalenol production. An RNA sequencing (RNA-seq) analysis indicated that FgNhp6 deletion influenced a wide array of metabolic pathways, particularly affecting several secondary metabolic pathways, such as sterol biosynthesis and aurofusarin biosynthesis. The findings of this study highlight the essential role of FgNhp6 in the regulation of the asexual and sexual reproduction, deoxynivalenol (DON) production, and pathogenicity of F. graminearum.

Structure and Functions of HMGB3 Protein.

HMGB3 protein belongs to the group of HMGB proteins from the superfamily of nuclear proteins with high electrophoretic mobility. HMGB proteins play an active part in almost all cellular processes associated with DNA-repair, replication, recombination, and transcription-and, additionally, can act as cytokines during infectious processes, inflammatory responses, and injuries. Although the structure and functions of HMGB1 and HMGB2 proteins have been intensively studied for decades, very little attention has been paid to HMGB3 until recently. In this review, we summarize the currently available data on the molecular structure, post-translational modifications, and biological functions of HMGB3, as well as the possible role of the ubiquitin-proteasome system-dependent HMGB3 degradation in tumor development.

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.

Systematic analysis of expression profiles of HMGB family members for prognostic application in non-small cell lung cancer.

Background: Lung cancer is a significant challenge to human health. Members of the high mobility group (HMG) superfamily (HMGB proteins) are implicated in a wide variety of physiological and pathophysiological processes, but the expression and prognostic value of HMGB family members in non-small cell lung cancer (NSCLC) have not been elucidated. Methods: In this study, ONCOMINE, UALCAN, GEPIA, Kaplan–Meier Plotter, starBase, OncomiR databases, and GeneMANIA were utilized to evaluate the prognostic significance of HMGB family members in NSCLC. Results: HMGB2/3 expression levels were higher in NSCLC patients. HMGB1 expression was higher in lung squamous cell carcinoma (LUSC) and was lower in lung adenocarcinoma (LUAD) tissue than in normal lung tissue. HMGB2 expression was related to cancer stage. Increased HMGB1 mRNA expression levels were associated with improved lung cancer prognosis, including overall survival (OS), first-progression survival (FP), and post-progression survival (PPS). There was no significant association between HMGB2 levels and prognostic indicators. HMGB3 expression was associated with poorer OS. GeneMANIA and GO/KEGG pathway analysis showed that HMGB family members mainly associated with chromosome condensation, regulation of chromatin organization, and nucleosome binding in NSCLC. HMGBs expression were closely correlated with infiltrating levels of specific types of immune cells in NSCLC, especially Th2 cells, Th17 cells, and mast cells. hsa-miR-25-3p, hsa-miR-374a-3p, and hsa-miR-93-5p were significantly positively correlated with HMGB1, HMGB2, and HMGB3, respectively. However, hsa-miR-30a-5p was predicted to significantly negatively regulate HMGB3 expression. Conclusion: Our study revealed that HMGB1 is positively related to the improved prognosis in NSCLC, and demonstrate that HMGB3 might be a risk factor for poorer survival of NSCLC patients.

Abstract 1512: Architectural protein HMGB3 interacts with cisplatin-resistance associated overexpressed protein (CROP/LUC7L3) in human cancer cells and modulates cisplatin-DNA adduct removal

Abstract Cancer treatment and management have improved substantially in recent years resulting in increased overall patient survival. However, upon recurrence, many tumors can eventually stop responding to treatment and become resistant to therapy. Therefore, sensitizing cancer cells to therapy is an important approach to increase overall patient survival and improve their quality of life. We have previously shown that targeting the chromatin associated architectural protein High Mobility Group Box 3 (HMGB3) can sensitize cisplatin resistant human ovarian cancer cells to cisplatin. HMGB proteins are non-histone chromatin proteins, which bind to distorted DNA structures with higher affinity compared to double-stranded DNA. HMGB proteins have been shown to play important roles in DNA metabolism, such as replication, transcription and repair. We found that the sensitization of cisplatin resistant human A2780/CP70 ovarian cancer cells by siRNA-mediated targeting of HMGB3 is achieved, at least in part, by significantly lowering the expression of the DNA damage response (DDR) proteins ATR and CHK1. In addition, depletion of HMGB3 repressed global transcription by ~50% in human osteosarcoma (U2OS) cells. Here we report that GFP-tagged HMGB3 protein forms distinct foci in human A2780 ovarian cancer cells upon cisplatin treatment. Further, depleting HMGB3 in cisplatin resistant A2780/CP70 cells reduces the rate of cisplatin-DNA adduct removal, suggesting a possible role in chemoresistance. In addition, mass spectrometry studies reveal that HMGB3 interacts with the cisplatin resistance-associated overexpressed protein (CROP, also known as LUC7L3 ), following treatment with the DNA interstrand crosslink-inducing agent, psoralen (plus UVA irradiation). LUC7L3 is associated with the spliceosome and is overexpressed in A2780/CP70 cells compared to A2780 ovarian cancer cells. SiRNA-mediated depletion of HMGB3 reduces the transcription levels of LUC7L3. Further, siRNA-mediated depletion of LUC7L3 reduced the clone forming capacity of U2OS cells and ovarian cancer cells. Analysis of cancer patient databases revealed that LUC7L3 mRNA is overexpressed in many cancer types including ovarian cancer. Further analysis showed that ovarian cancer, gastric cancer, liver cancer and lung cancer patients overexpressing LUC7L3 have a significantly reduced overall survival than those with lower levels. Taken together, these data indicate that in addition to the architectural roles of HMGB3, which is important for DNA metabolism, transcriptional modulation of DDR proteins through interaction with the spliceosome may be another way that HMGB3 modulates chemotherapeutic DNA damage processing in human cancer cells. Citation Format: Anirban Mukherjee, Lorraine-Rana Benhamou, Karen M. Vasquez. Architectural protein HMGB3 interacts with cisplatin-resistance associated overexpressed protein (CROP/LUC7L3) in human cancer cells and modulates cisplatin-DNA adduct removal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1512.

Mechanisms coordinating ribosomal protein gene transcription in response to stress.

While expression of ribosomal protein genes (RPGs) in the budding yeast has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating growth conditions. Most RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs) and are further differentiated by the presence or absence of the HMGB protein Hmo1. However, a third group of promoters appears not to be bound by any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate here that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated downregulation upon stress. At the majority of RPG promoters, Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected protein homeostasis-dependent binding property of Hmo1 at RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator. Our study provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.

The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA.

DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

FTIR Study of the Secondary Structure of DNA in Complexes with Platinum Coordination Compounds

Mechanisms of interaction between antitumor compounds and biological molecules have been being studied for decades. One of the most widely applicable drug is dichlorodiammineplatinum(II) (DDP). In the present work we have analyzed effect of cis- and trans-DDP on secondary structure of DNA, bovine serum albumins and HMGB proteins using FTIR spectroscopy. We have shown that both of the DDP isomers facilitate the formation of albumin dimers. We have also shown that DDP facilitates changes in DNA structure, attributed to DDP binding to DNA bases and DNA cross-linking. Thus, the application of hidden peak analysis of FTIR spectra shown to be informative to structural investigation of DDP complexes with nucleic acids and proteins.

Targeting the High-Mobility Group Box 3 Protein Sensitizes Chemoresistant Ovarian Cancer Cells to Cisplatin.

Chemotherapeutic regimens for ovarian cancer often include the use of DNA interstrand crosslink-inducing agents (e.g., platinum drugs) or DNA double-strand break-inducing agents. Unfortunately, the majority of patients fail to maintain a durable response to treatment, in part, due to drug resistance, contributing to a poor survival rate. In this study, we report that cisplatin sensitivity can be restored in cisplatin-resistant ovarian cancer cells by targeting the chromatin-associated high-mobility group box 3 (HMGB3) protein. HMGB proteins have been implicated in the pathogenesis and prognosis of ovarian cancer, and HMGB3 is often upregulated in cancer cells, making it a potential selective target for therapeutic intervention. Depletion of HMGB3 in cisplatin-sensitive and cisplatin-resistant cells resulted in transcriptional downregulation of the kinases ATR and CHK1, which attenuated the ATR/CHK1/p-CHK1 DNA damage signaling pathway. HMGB3 was associated with the promoter regions of ATR and CHK1, suggesting a new role for HMGB3 in transcriptional regulation. Furthermore, HMGB3 depletion significantly increased apoptosis in cisplatin-resistant A2780/CP70 cells after cisplatin treatment. Taken together, our results indicate that targeted depletion of HMGB3 attenuates cisplatin resistance in human ovarian cancer cells, increasing tumor cell sensitivity to platinum drugs. SIGNIFICANCE: This study shows that targeting HMGB3 is a potential therapeutic strategy to overcome chemoresistance in ovarian cancer.

Roles of High Mobility Group Box Proteins in Nucleotide Excision Repair‐associated Processing of DNA Interstrand Crosslinks

Many traditional anti‐cancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs), lesions that form covalent bonds between the DNA strands. ICLs are highly cytotoxic if not properly repaired because they can block basic cellular processes such as transcription and replication. Nucleotide excision repair (NER) is a critical repair mechanism in processing ICLs in human cells. In addition to NER, ICLs are also processed through DNA double‐strand break repair pathways (e.g. homologous recombination) in S‐phase. We have previously demonstrated that the high mobility group box protein (HMGB1), a non‐histone architectural protein, can bind to site‐directed ICLs with high affinity and modulate error‐free repair processing of such lesions. In addition, we have found that HMGB1 associates with the NER damage recognition protein XPA in human cells and facilitates the recruitment of XPA to the damaged site. Further, we have demonstrated that HMGB1 can architecturally modify the damaged substrate by inducing negative supercoils, which may facilitate DNA damage processing by NER. We hypothesize that HMGB1 can modulate NER processing of ICLs by aiding NER damage‐recognition factors in locating damaged DNA. To test this hypothesis we are using a mutation‐reporter system to measure mutation frequencies and spectra generated by site‐directed ICLs in human cells. There are two NER subpathways that recognize DNA damage via different mechanisms. I am investigating in which pathway(s) HMGB1 is involved by utilizing various cell lines that are proficient or deficient for an essential recognition protein in each pathway. Additionally, we are investigating if HMGB2 and HMGB3, protein family members that are highly similar in structure to HMGB1, are also co‐factors in NER. Preliminary evidence suggests that HMGB1 may be involved in transcription coupled‐NER of site‐directed ICLs. If future evidence supports these data, this would indicate that HMGB1 could associate with transcription machinery and aid in recruiting other NER co‐factors, such as XPA. Future genetic, molecular biological, and biochemical assays will provide additional insight as to the roles of the HMGB proteins in ICL repair. The processing and repair of ICLs in human cells is not completely understood, and therefore a detailed study to define the molecular mechanisms of ICL processing is warranted. Identifying new proteins involved in ICL processing may lead to new pharmacological targets, which may help us to improve the outcome of cancer treatment.Support or Funding InformationNIH/NCI to K.V. (CA093729) and NSF GRFP to J.G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Destabilizing Nucleosomes and the Role of HMGB Proteins

Destabilizing Nucleosomes and the Role of HMGB Proteins

Cloning and characterization of the LEF/TCF gene family in grass carp (Ctenopharyngodon idella) and their expression profiles in response to grass carp reovirus infection

T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins from the High Mobility Group (HMG) box family act as the main downstream effectors of the Wnt signaling pathway. HMGB proteins play multifaceted roles in the immune system of mammals. To clarify the immunological characteristics of LEF/TCF genes in grass carp (Ctenopharyngodon idella), five LEF/TCF genes (TCF7, LEF1, TCF7L1A, TCF7L1B, and TCF7L2) were identified and characterized. All five LEF/TCF proteins contained two characteristic domains: a HMG-BOX domain and a CTNNB1_binding region. Phylogenetic tree analysis revealed that the LEF/TCF proteins were represented different lineages. These results of subcellular localization showed that four of the LEF/TCF genes were localized exclusively within the nucleus, while TCF7L2 was localized in the cytoplasm and nucleus. The mRNA expression profiles of these LEF/TCF family genes differed across different tissues. The mRNA expression levels of TCF7, TCF7L1A, and TCF7L2 changed significantly in liver after grass carp reovirus (GCRV) challenge; TCF7 and TCF7L1A responded early while TCF7L2 responded late. This suggests that these genes may participate in GCRV-related immune responses. Moreover, TCF7 promoted Bcl6 transcription in response to the GCRV challenge. These findings further our understanding of the function of LEF/TCF genes in teleosts.

HMGB Proteins as DNA Chaperones That Modulate Chromatin Activity

HMGB Proteins as DNA Chaperones That Modulate Chromatin Activity

Abstract 1375: High mobility group box proteins modulate chemotherapeutic DNA damage repair processing in human cells

Abstract Many anticancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs) and DNA double-strand breaks (DSBs) leading to cytotoxicity; however, the mechanisms involved in processing such damage is not completely understood. Thus, a better understanding of the processing of ICLs and DSBs will assist in the identification of novel pharmacological targets, and improved drug design, particularly in chemo-resistant populations. We discovered that the High Mobility Group Box proteins (HMGBs), a family of non-histone architectural proteins, modulate DNA lesion processing in human cancer cells and significantly alter cell survival. We have shown that one of the HMGB family members, HMGB1, binds with high affinity to ICLs targeted to specific sites using triplex-forming oligonucleotides (TFOs), and modulates the repair of ICLs as a co-factor of the nucleotide excision repair (NER) mechanism. We found that other HMGB family members, HMGB2 and HMGB3, which share sequence and structural homology with HMGB1, also modulate DNA repair. Based on our preliminary results, we hypothesize that the HMGB proteins modulate lesion processing via unique and distinct mechanisms, and promote the repair of chemotherapeutic and carcinogenic lesions. In our studies site-specific ICLs were formed by incubating DNA substrates with psoralen-conjugated TFOs followed by UVA irradiation (TFO-ICLs) and used in mutagenesis studies in human cells. Protein recruitment to the damaged site was determined by chromatin immuno-precipitation (ChIP). Non-homologous end-joining (NHEJ) and homologous recombination (HR) were studied via fluorescence activated cell sorting of fluorescent reporter genes in human cells. Cellular localization was visualized by immunofluorescence microscopy. Chromosomal aberrations were determined from metaphase spreads using light microscopy. In response to ICLs, siRNA-mediated depletion of HMGB proteins increased mutagenesis, suggesting involvement in error-free DNA damage repair. Interestingly, analysis of the mutants indicated an increase in single nucleotide mutations in the absence of HMGB1, whereas depletion of HMGB2 or HMGB3 predominantly resulted in small deletions, indicating their involvement in different repair pathways. Specifically, HMGB2 or HMGB3 depletion reduced ICL-induced single-strand annealing (a sub-pathway of HR) events. Interestingly, we found that both HMGB1 and HMGB2 forms distinct foci after damage induction. HMGB2 depletion stimulated NHEJ events, while depletion of HMGB2 or HMGB3 reduced DSB-induced HR events. Finally, depletion of HMGB proteins increased chromosomal aberrations in cancer cells treated with ICL- and DSB-inducing agents. Our novel findings suggest that the HMGB proteins are involved in processing of carcinogenic and chemotherapeutic lesions in human cells via distinct repair mechanisms. Citation Format: Anirban Mukherjee, Wade A. Reh, Karen M. Vasquez. High mobility group box proteins modulate chemotherapeutic DNA damage repair processing in human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1375.

Ixr1 Regulates Ribosomal Gene Transcription and Yeast Response to Cisplatin

Ixr1 is a Saccharomyces cerevisiae HMGB protein that regulates the hypoxic regulon and also controls the expression of other genes involved in the oxidative stress response or re-adaptation of catabolic and anabolic fluxes when oxygen is limiting. Ixr1 also binds with high affinity to cisplatin-DNA adducts and modulates DNA repair. The influence of Ixr1 on transcription in the absence or presence of cisplatin has been analyzed in this work. Ixr1 regulates other transcriptional factors that respond to nutrient availability or extracellular and intracellular stress stimuli, some controlled by the TOR pathway and PKA signaling. Ixr1 controls transcription of ribosomal RNAs and genes encoding ribosomal proteins or involved in ribosome assembly. qPCR, ChIP, and 18S and 25S rRNAs measurement have confirmed this function. Ixr1 binds directly to several promoters of genes related to rRNA transcription and ribosome biogenesis. Cisplatin treatment mimics the effect of IXR1 deletion on rRNA and ribosomal gene transcription, and prevents Ixr1 binding to specific promoters related to these processes.

The HMGB protein Ixr1 interacts with Ssn8 and Tdh3 involved in transcriptional regulation.

Ixr1 is a Saccharomyces cerevisiae transcriptional factor that extensively regulates the response to hypoxia and controls other important cellular functions and DNA repair. During aerobic growth, the Ixr1 repressor function is predominant on regulated promoters of hypoxic genes, although activator effects are also observed on other genes. During hypoxia, Ixr1 expression increases and the number of genes activated by Ixr1 also increase. In this work we demonstrate that the NH2-terminal region of Ixr1 is involved in transcriptional activation. We also present the first analysis about Ixr1 interactions with three factors that have been previously identified as important players in the yeast hypoxic response, Cyc8, Tup1 and Ssn8; results demonstrate that only Ssn8 binds to Ixr1. We have also looked for other Ixr1-binding proteins associated with transcriptional regulation, by co-purification and mass spectrometry identification. Tdh3, a protein involved in transcriptional silencing, is among the new identified Ixr1-binding proteins. Differential phosphorylation of Ixr1 is found when comparing aerobic and hypoxic yeast growth. Implication of these results in transcriptional regulation mediated by Ixr1 is discussed.

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.

Using Chromatin Conformation Capture (3C) to Investigate Length-Dependent Saccharomyces Cerevisiae Gene Looping with and without HMGB Proteins

HMGB proteins are able to enhance the apparent flexibility of DNA, presumably through the creation of flexible kinks in the DNA. These sequence non-specific proteins may facilitate chromatin looping and dynamics by acting on the rigid segments of inter-nucleosomal linker DNA and might affect chromatin stability by directly interacting with nucleosomes. We are interested in understanding how loss of HMGB proteins in yeast affects the ability of chromatin to form loops over the length scale of full yeast genes (several thousand base pairs). We hypothesize that gene looping over this scale will be inhibited in a length-dependent manner in Saccharomyces cerevisiae strains disrupted for NHP6A/B, NHP10, or HMO1 genes encoding sequence non-specific HMGB proteins. We have engineered these gene disruptions in yeast and have adapted the conventional Chromatin Conformation Capture (3C) assay for quantitative PCR analysis to compare gene looping in wild-type versus mutant strains. Using this system with suitable gene loops, the yeast BLM10 gene that was recently shown to have a clear 3C looping signal in vivo as well as the yeast GAL10 and HEM3 genes, we report progress to date on testing the effects of HMGB deletion on gene looping. These data address the issue of how the intrinsic physical properties of DNA are managed in a biological context.

HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

Deciphering the role of the AT-rich interaction domain and the HMG-box domain of ARID-HMG proteins of Arabidopsis thaliana.

ARID-HMG DNA-binding proteins represent a novel group of HMG-box containing protein family where the AT-rich interaction domain (ARID) is fused with the HMG-box domain in a single polypeptide chain. ARID-HMG proteins are highly plant specific with homologs found both in flowering plants as well as in moss such as Physcomitrella. The expression of these proteins is ubiquitous in plant tissues and primarily localises in the cell nucleus. HMGB proteins are involved in several nuclear processes, but the role of ARID-HMG proteins in plants remains poorly explored. Here, we performed DNA-protein interaction studies with Arabidopsis ARID-HMG protein HMGB11 (At1g55650) to understand the functionality of this protein and its individual domains. DNA binding assays revealed that AtHMGB11 can bind double-stranded DNA with a weaker affinity (Kd = 475 ± 17.9nM) compared to Arabidopsis HMGB1 protein (Kd = 39.8 ± 2.68nM). AtHMGB11 also prefers AT-rich DNA as a substrate and shows structural bias for supercoiled DNA. Molecular docking of the DNA-AtHMGB11 complex indicated that the protein interacts with the DNA major groove, mainly through its ARID domain and the junction region connecting the ARID and the HMG-box domain. Also, predicted by the docking model, mutation of Lys(85) from the ARID domain and Arg(199) & Lys(202) from the junction region affects the DNA binding affinity of AtHMGB11. In addition, AtHMGB11 and its truncated form containing the HMG-box domain can not only promote DNA mini-circle formation but are also capable of inducing negative supercoils into relaxed plasmid DNA suggesting the involvement of this protein in several nuclear events. Overall, the study signifies that both the ARID and the HMG-box domain contribute to the optimal functioning of ARID-HMG protein in vivo.