Abundant Chromatin Protein Research Articles

Fusion Dynamics and Size-Dependence of Droplet Microstructure in ssDNA-Mediated Protein Phase Separation.

Biomolecular condensation involving proteins and nucleic acids has been recognized to play crucial roles in genome organization and transcriptional regulation. However, the biophysical mechanisms underlying the droplet fusion dynamics and microstructure evolution during the early stage of liquid-liquid phase separation (LLPS) remain elusive. In this work, we study the phase separation of linker histone H1, which is among the most abundant chromatin proteins, in the presence of single-stranded DNA (ssDNA) capable of forming a G-quadruplex by using molecular simulations and experimental characterization. We found that droplet fusion is a rather stochastic and kinetically controlled process. Productive fusion events are triggered by the formation of ssDNA-mediated electrostatic bridges within the droplet contacting zone. The droplet microstructure is size-dependent and evolves driven by maximizing the number of electrostatic contacts. We also showed that the folding of ssDNA to the G-quadruplex promotes LLPS by increasing the multivalency and strength of protein-DNA interactions. These findings provide deep mechanistic insights into the growth dynamics of biomolecular droplets and highlight the key role of kinetic control during the early stage of ssDNA-protein condensation.

PO-307 Paradoxical decrease in H2AFX copy number yet increasing mRNA expression in human breast cancer

IntroductionHistones are basic proteins responsible for DNA packaging in eukaryotic cell nuclei. H2AX is an H2A variant that plays an important role early in the DNA damage response. Although H2AX expression has been investigated, the direct contribution of this abundant chromatin protein in breast cancer is not well understood. The H2AFX gene encodes the same H2AX protein by both a short non-polyadenylated transcript and a longer polyadenylated transcript. We are characterising H2AFX copy number (CN), mRNA expression and protein abundance in breast cancer to discover if this foundation chromatin component contributing to genome stability could play a role in breast cancer.Material and methodsH2AFX mRNA abundance was analysed in a panel of breast cell lines by absolute quantitative PCR. The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset was used for bioinformatic analysis.Results and discussionsAnalysis of the METABRIC dataset shows that H2AFX copy number alterations involving loss of one allele are very common (41%) and correlate with genome instability. No diploid deletions are observed, and only 2% of samples show H2AFX CN amplifications. No mutations were observed in the H2AFX coding region. Although we observed that the H2AFX short transcript is more abundant than the long transcript in the panel of breast cell lines, we found that the ratio of short and long transcript is constant. This validates the relevance of transcriptomic databases using polyA-selected H2AFX mRNAs that only report long transcript abundance. Analysis of H2AFX mRNA levels in METABRIC revealed an increase in H2AFX expression with worsening prognosis, despite the decrease in gene dosage. Furthermore, we found that H2AFX copy number decreases while H2AFX mRNA expression increases during the progression from luminal A to luminal B which are the most common breast cancer subtypes.ConclusionAnalysis of primary breast tumour genomic datasets revealed that H2AFX frequently has reduced copy number but is almost never fully deleted, mutated, or amplified. One copy is lost usually as a result of a major deletion in the 11q chromosome arm. Paradoxically, H2AFX expression is frequently increased suggesting that an independent mechanism for regulation exists to drive up-regulation. We are now bringing together H2AFX copy number, mRNA expression and protein data to understand whether H2AFX expression responds to challenges to genome integrity in breast cancer.

Expression of HMGB2 indicates worse survival of patients and is required for the maintenance of Warburg effect in pancreatic cancer.

High mobility group proteins (HMGs) are the second most abundant chromatin proteins and exert global genomic functions in the establishment of active or inactive chromatin domains. Through interaction with nucleosomes, transcription factors, nucleosome-remodeling machines and histones, the HMGs family proteins contribute to the fine tuning of transcription in response to rapid environmental changes. Mammalian high mobility group Bs (HMGBs) are characterized by two tandem HMG box domains followed by a long acidic tail. Recent studies demonstrated that high expression of HMGBs has been found in many cancers, such as prostate, kidney, ovarian, and gastric cancers. However, their roles in pancreatic cancer have seldom been reported. In this study, we assessed the diagnostic and prognostic values of HMGBs proteins, including HMGB1, HMGB2, and HMGB3, in pancreatic cancer from the Cancer Genome Atlas (TCGA) dataset. Our results demonstrated that HMGB2 predicted poor prognosis in pancreatic cancer. In vitro studies demonstrated that silencing HMGB2 inhibited cell proliferation and viability. Mechanistically, our results demonstrated that silencing HMGB2 decreased hypoxia inducible factor 1α (HIF1α) protein level and inhibited HIF1α-mediated glycolysis process. Further analysis indicated that HIF1α-targeted glycolytic genes, including GLUT1, HK2, and LDHA, are all prognostic factors and positively correlated with HMGB2 expression. Taken together, we discovered new prognostic and predictive markers for pancreatic cancer, and shed light on the novel function of HMGB2 in glycolytic control in cancer.

Identification and characterization of nonhistone chromatin proteins: human positive coactivator 4 as a candidate.

The highly dynamic nucleoprotein structure of eukaryotic genome is organized in an ordered fashion, the unit of which is the nucleosome. The nucleosome is composed of core histones and DNA of variable size wrapped around it. Apart from the histone proteins, several nonhistone proteins also interact with the complex consisting of the DNA, the core and linker histones conferring highly regulated fluidity on the chromatin and permitting fine tuning of its functions. The nonhistone proteins are multifunctional and accentuate diverse cellular outcomes. In spite of the technical challenges, the architectural role of the nonhistone proteins altering the topology of the chromatin has been studied extensively. To appreciate the significance of the chromatin for genome function, it is essential to examine the role of the nonhistone proteins in different physiological conditions. Here, taking the example of a highly abundant chromatin protein, PC4 (Positive coactivator 4), we describe strategies for the identification of the chromatin-associated proteins and their structural and functional characterization.

Topoisomerase II: a fitted mechanism for the chromatin landscape

The mechanism by which type-2A topoisomerases transport one DNA duplex through a transient double-strand break produced in another exhibits fascinating traits. One of them is the fine coupling between inter-domainal movements and ATP usage; another is their preference to transport DNA in particular directions. These capabilities have been inferred from in vitro studies but we ignore their significance inside the cell, where DNA configurations markedly differ from those of DNA in free solution. The eukaryotic type-2A enzyme, topoisomerase II, is the second most abundant chromatin protein after histones and its biological roles include the decatenation of newly replicated DNA and the relaxation of polymerase-driven supercoils. Yet, topoisomerase II is also implicated in other cellular processes such as chromatin folding and gene expression, in which the topological transformations catalysed by the enzyme are uncertain. Here, some capabilities of topoisomerase II that might be relevant to infer the enzyme performance in the context of chromatin architecture are discussed. Some aspects addressed are the importance of the DNA rejoining step to ensure genome stability, the regulation of the enzyme activity and of its putative structural role, and the selectively of DNA transport in the chromatin milieu.

Antagonizing HMGB1 inhibits proteinuria in a murine model of lupus-like disease (131.24)

Abstract High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts like an inflammatory cytokine when it is released by dying or stimulated cells. Elevated levels are detected at sites of inflammation in a number of autoimmune disorders including SLE. In addition, HMGB1 has been shown to contribute to the activation of plasmacytoid dendritic cells as well as B cells, cell types that are implicated in the pathogenesis of SLE. To address the role of HMGB1 in experimental lupus, we investigated the protective efficacy of a fully humanized mAb (IA-4, which recognizes both mouse and human HMGB1) in an IFNα-accelerated lupus model. In this model, adenovirus IFNα-treated NZB/W F1 mice exhibit 90–100% proteinuria incidence at 4–6 weeks post-treatment. The functionality of IA-4 has been demonstrated in vitro by its ability to inhibit rHMGB1-induced IL-6 secretion in human PBMC (IC50 = 0.9 nM). Prophylactic administration of the anti-HMGB1 mAb IA-4 at 10 mg/kg significantly inhibited the proteinuria severity score at seven weeks (65% inhibition; p<0.05 compared to an isotype control). Consistent with the proteinuria data, histopathological assessment of the kidney also revealed protection with IA-4 treatment. Despite its protection in nephritis, IA-4 treatment showed no detectable effect on serum levels of IFN-α and autoantibodies against dsDNA or SSA/Ro. Taken together, these data suggest that HMGB1 is a critical effector molecule in lupus nephritis and that anti-HMGB1 treatment may provide a potential therapeutic benefit for the treatment of SLE.

CC1, a novel crenarchaeal DNA binding protein.

The genomes of the related crenarchaea Pyrobaculum aerophilum and Thermoproteus tenax lack any obvious gene encoding a single-stranded DNA binding protein (SSB). SSBs are essential for DNA replication, recombination, and repair and are found in all other genomes across the three domains of life. These two archaeal genomes also have only one identifiable gene encoding a chromatin protein (the Alba protein), while most other archaea have at least two different abundant chromatin proteins. We performed a biochemical screen for novel nucleic acid binding proteins present in cell extracts of T. tenax. An assay for proteins capable of binding to a single-stranded DNA oligonucleotide resulted in identification of three proteins. The first protein, Alba, has been shown previously to bind single-stranded DNA as well as duplex DNA. The two other proteins, which we designated CC1 (for crenarchaeal chromatin protein 1), are very closely related to one another, and homologs are restricted to the P. aerophilum and Aeropyrum pernix genomes. CC1 is a 6-kDa, monomeric, basic protein that is expressed at a high level in T. tenax. This protein binds single- and double-stranded DNAs with similar affinities. These properties are consistent with a role for CC1 as a crenarchaeal chromatin protein.

The SAF-box domain of chromatin protein DEK

DEK is an abundant chromatin protein in metazoans reaching copy numbers of several millions/nucleus. Previous work has shown that human DEK, a protein of 375 amino acids, has two functional DNA-binding domains, of which one resides in a central part of the molecule and contains sequences corresponding to the scaffold attachment factor-box (SAF-box) domain as found in a growing number of nuclear proteins. Isolated SAF-box peptides (amino acids 137–187) bind weakly to DNA in solution, but when many SAF-box peptides are brought into close proximity on the surface of Sephadex beads, cooperative effects lead to a high affinity to DNA. Furthermore, a peptide (amino acids 87–187) that includes a sequence on the N-terminal side of the SAF-box binds efficiently to DNA. This peptide prefers four-way junction DNA over straight DNA and induces supercoils in relaxed circular DNA just like the full-length DEK. Interestingly, however, the 87–187 amino acid peptide introduces negative supercoils in contrast to the full-length DEK, which is known to introduce positive supercoils. We found that two adjacent regions (amino acids 68–87 and 187–250) are necessary for the formation of positive supercoils. Our data contribute to the ongoing characterization of the abundant and ubiquitous DEK chromatin protein.

Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration and proliferation.

High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Here, we show that extracellular HMGB1 and its receptor for advanced glycation end products (RAGE) induce both migration and proliferation of vessel-associated stem cells (mesoangioblasts), and thus may play a role in muscle tissue regeneration. In vitro, HMGB1 induces migration and proliferation of both adult and embryonic mesoangioblasts, and disrupts the barrier function of endothelial monolayers. In living mice, mesoangioblasts injected into the femoral artery migrate close to HMGB1-loaded heparin-Sepharose beads implanted in healthy muscle, but are unresponsive to control beads. Interestingly, α-sarcoglycan null dystrophic muscle contains elevated levels of HMGB1; however, mesoangioblasts migrate into dystrophic muscle even if their RAGE receptor is disabled. This implies that the HMGB1–RAGE interaction is sufficient, but not necessary, for mesoangioblast homing; a different pathway might coexist. Although the role of endogenous HMGB1 in the reconstruction of dystrophic muscle remains to be clarified, injected HMGB1 may be used to promote tissue regeneration.

Chromatin Structure in Granulocytes: A LINK BETWEEN TIGHT COMPACTION AND ACCUMULATION OF A HETEROCHROMATIN-ASSOCIATED PROTEIN (MENT)

To study the mechanism of heterochromatin formation in vertebrate cells, we isolated nuclei from chicken polymorphonuclear granulocytes and examined the chromatin organization. We found granulocyte chromatin to remain insoluble after nuclease digestion and to be resistant to swelling in low salt/high pH media. Both insolubility and resistance to swelling were lost after washing with 0.3 M NaCl, a procedure that released two abundant tissue-specific proteins from granulocyte nuclei. One of them (42 kDa) is identified as MENT, a protein previously shown to be associated with repressed chromatin from mature chicken erythrocytes. We show that MENT is immunolocalized in granulocyte heterochromatin, where it is one of the most abundant chromatin proteins ( approximately 2 molecules/200 base pairs of DNA). MENT is the first nuclear protein structurally related to the serine protease inhibitor family. The other abundant protein is similar to or identical with mim-1, a myeloid-specific protein that is known to be stored in cell granules and to associate with isolated nuclei. MENT (but not mim-1) binds chromatin and free DNA, and, at its physiological protein/DNA ratio, enhances compaction and the reversible Mg2+-dependent self-association of nucleosome arrays. MENT appears to promote the formation of heterochromatin by acting as a "glue" within and between chains of nucleosomes.

The C-terminal domain of SIN1 in yeast interacts with a protein that binds the URS1 region of the yeast HO gene.

A protein or protein complex has previously been identified in Saccharomyces cerevisiae which both binds a short DNA sequence in URS1 of HO and interacts with SIN1. SIN1, which has some sequence similarity to mammalian HMG1, is an abundant chromatin protein in yeast and is thought to participate in the transcriptional repression of a specific family of genes. SIN1 binds DNA weakly, though it has no DNA binding specificity. Here we address the nature of the interaction between SIN1 and the specific DNA binding protein(s) to HO DNA. We show that the isolated C-terminal region of SIN1 can interact in vitro with the DNA binding protein, causing a supershift in a gel mobility shift assay. Interestingly, inclusion of the region in SIN1 which contains two acidic sequences, precludes the binding of recombinant protein to the DNA/protein complex.

Altered chromatin structure of cerebral nuclei in experimental diabetes mellitus.

To determine whether diabetes alters chromatin structure in vivo, micrococcal nuclease digestion kinetics were analyzed in cerebral cortical and hepatic nuclei of streptozotocin-induced diabetic rats. Cerebral nuclei of diabetic rats maintained for 6 weeks were less susceptible to micrococcal nuclease digestion compared with control rats. Insulin treatment reversed diabetes-related changes in nuclease digestion kinetics. There were no changes in the kinetics of digestion in hepatic nuclei. The reduced digestibility of cerebral DNA in diabetes could not be attributed to altered DNA fluorescence spectra, or altered distribution of most abundant chromatin proteins that were either solubilized or that remained insoluble immediately following nuclease digestion. It is concluded that chronic, uncontrolled hyperglycemia can alter chromatin structure of some tissues in vivo, and this change is probably related to subtle alterations in DNA-protein interactions.