Chaperoning of the histone octamer by the acidic domain of DNA repair factor APLF

Nucleosome assembly requires the coordinated deposition of histone complexes H3-H4 and H2A-H2B to form a histone octamer on DNA. In the current paradigm, specific histone chaperones guide the deposition of first H3-H4 and then H2A-H2B(1-5). Here, we show that the acidic domain of DNA repair factor APLF (APLFAD) can assemble the histone octamer in a single step, and deposit it on DNA to form nucleosomes. The crystal structure of the APLFAD-histone octamer complex shows that APLFAD tethers the histones in their nucleosomal conformation. Mutations of key aromatic anchor residues in APLFAD affect chaperone activity in vitro and in cells. Together, we propose that chaperoning of the histone octamer is a mechanism for histone chaperone function at sites where chromatin is temporarily disrupted.

The polyanionic drug suramin neutralizes histones and prevents endotheliopathy

Drugs are needed to protect against the neutrophil derived histones responsible for endothelial injury in acute inflammatory conditions such as trauma and sepsis. Heparin and other polyanions can neutralize histones but may cause secondary, deleterious effects such as excessive bleeding. Here, we demonstrate that suramin (a widely available polyanionic drug) completely neutralizes the toxic effects of histones. The sulfate groups on suramin form stable electrostatic interactions with hydrogen bonds in the histone octamer with a dissociation constant of 250 nM. In cultured endothelial cells (Ea.Hy926), histone induced thrombin generation was significantly decreased by suramin. In isolated murine blood vessels, suramin abolished aberrant endothelial cell calcium signals and rescued impaired endothelial dependent vasodilation caused by histones. Suramin significantly decreased pulmonary endothelial cell ICAM-1 expression and neutrophil recruitment caused by infusion of sublethal doses of histones in vivo. Suramin also prevented lung edema, intraalveolar hemorrhage and mortality in mice receiving a lethal dose of histones. Protection of vascular endothelial function from histone-induced damage is a novel mechanism of action for suramin with therapeutic implications for conditions characterized by elevated histone levels.

Characterizing circulating nucleosomes in the plasma of dogs with lymphoma

Background Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. Results Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. Conclusions The Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines.

Structures and implications of TBP–nucleosome complexes

The TATA box-binding protein (TBP) is highly conserved throughout eukaryotes and plays a central role in the assembly of the transcription preinitiation complex (PIC) at gene promoters. TBP binds and bends DNA, and directs adjacent binding of the transcription factors TFIIA and TFIIB for PIC assembly. Here, we show that yeast TBP can bind to a nucleosome containing the Widom-601 sequence and that TBP–nucleosome binding is stabilized by TFIIA. We determine three cryo-electron microscopy (cryo-EM) structures of TBP–nucleosome complexes, two of them containing also TFIIA. TBP can bind to superhelical location (SHL) –6, which contains a TATA-like sequence, but also to SHL +2, which is GC-rich. Whereas binding to SHL –6 can occur in the absence of TFIIA, binding to SHL +2 is only observed in the presence of TFIIA and goes along with detachment of upstream terminal DNA from the histone octamer. TBP–nucleosome complexes are sterically incompatible with PIC assembly, explaining why a promoter nucleosome generally impairs transcription and must be moved before initiation can occur.

Characterizing circulating nucleosomes in the plasma of dogs with hemangiosarcoma

Background Nucleosomes consist of DNA wrapped around a histone octamer core like thread on a spool to condense DNA as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death, chromatin fragmentation and release of nucleosomes into the blood. The Nu.Q™ platform measures circulating nucleosomes in the blood of humans that result from disease and has been used to detect and identify cancer even at early stages. The objectives of this study are to quantify and better characterize nucleosomes in dogs with various stages of hemangiosarcoma (HSA) using this ELISA-based assay. Samples from 77 dogs with a confirmed diagnosis of hemangiosarcoma and 134 healthy controls were utilized for this study. The HSA samples were recruited from the Texas A&M University Small Animal Clinic (TAMU-SAC) or purchased from biobanks. All control samples were recruited from the TAMU-SAC. Results Dogs with hemangiosarcoma had a 6.6-fold increase in their median plasma nucleosome concentrations compared to controls (AUC 92.9 %). Elevated nucleosome concentrations were seen at all stages of disease and nucleosome concentrations increased with the stage of the disease. Conclusions Plasma nucleosome concentrations are a reliable way to differentiate dogs with hemangiosarcoma from healthy dogs. Further testing is underway to better characterize cancer associated HSA circulating nucleosomes and optimize future diagnostics for canine HSA detection.

Structure and function of the chromatin remodeler SMARCAD1 with its nucleosome substrate

The ATP-dependent chromatin remodeler SMARCAD1 acts on nucleosomes during DNA repair and transcription, but despite its implication in disease, information on its structure and function is scarce. Chromatin remodelers use a variety of ways to engage nucleosomes, and outcomes of the ATP-dependent reactions vary widely. Here we show that SMARCAD1 transfers the entire histone octamer from one DNA segment to another in an ATP-dependent manner but is also capable of de novo nucleosome assembly from histone octamer, due to its ability to bind all histones simultaneously. We describe the cryoEM structure of SMARCAD1 in complex with a nucleosome and show that it engages its substrate unlike any other chromatin remodeler. Our combined data allow us to put forward a testable model for SMARCAD1 mechanism.One-Sentence SummaryThe single subunit chromatin remodeler SMARCAD1 engages nucleosomes in a unique manner and transfers the entire histone octamer.

Characterizing Circulating Nucleosomes in the Plasma of Dogs with Lymphoma

Background: Nucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.QTM H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay. A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU. Samples were also collected longitudinally from 3 dogs undergoing treatment for multicentric lymphoma at TAMU as a pilot study to investigate the pattern of nucleosome concentrations in plasma during treatment. Results: Dogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas. Nucleosome concentrations from serially monitored patients were elevated at diagnosis and progression with subsequent decreases in nucleosome concentration that corresponded to clinically detectable responses to therapy. Conclusions: The Nu.QTM H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines. Results from serially monitored patients indicate that nucleosomes could be an objective monitoring tool for remission status in canine lymphoma patients.

Reduce, Retain, Recycle: Mechanisms for Promoting Histone ProteinDegradation Versus Stability and Retention

The eukaryotic genome is packaged into chromatin. The nucleosome, the basic unit of chromatin, is composed of DNA coiled around a histone octamer. Histones are among the longest-lived protein species in mammalian cells, due to their thermodynamic stability and their associations with DNA and histone chaperones. Histone metabolism plays an integral role in homeostasis. While histones are largely stable, degradation of histone proteins is necessary under specific conditions. Here we review the physiological and cellular contexts which promote histone degradation. We describe specific known mechanisms that drive histone proteolysis. Finally, we discuss the importance of histone degradation and regulation of histone supply for organismal and cellular fitness.