Protein arginine N-methyltransferases are a family of epigenetic enzymes responsible for monomethylation or dimethylation of arginine residues on histones. Dysregulation of protein arginine N-methyltransferase activity can lead to aberrant gene expression and cancer. Recent studies have shown that PRMT2 expression and histone H3 methylation at arginine 8 are correlated with disease severity in glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma. In this study, we explore a noncatalytic mechanistic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histone peptides and proteins, and other PRMTs using analytical and enzymatic approaches. We quantify interactions between PRMT2, peptide ligands, and PRMT1 in a cofactor- and domain-dependent manner using differential scanning fluorimetry. We found that PRMT2 modulates the substrate specificity of PRMT1. Using calf thymus histones as substrates, we saw that a 10-fold excess of PRMT2 promotes PRMT1 methylation of both histone H4 and histone H2A. We found equimolar or a 10-fold excess of PRMT2 to PRMT1 can improve the catalytic efficiency of PRMT1 towards individual histone substrates H2A, H3, and H4. We further evaluated the effects of PRMT2 towards PRMT1 on unmodified histone octamers and mononucleosomes and found marginal PRMT1 activity improvements in histone octamers but significantly greater methylation of mononucleosomes in the presence of 10-fold excess of PRMT2. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in driving site-specific histone methylation marks.

Ubiquitin-specific peptidase 16 (USP16) is a deubiquitinase that plays a role in the regulation of gene expression, cell cycle progression, and various other functions. It was originally identified as the major deubiquitinase for histone H2A and has since been found to deubiquitinate a range of other substrates, including proteins from both the cytoplasm and nucleus. USP16 is phosphorylated when cells enter mitosis and dephosphorylated during the metaphase/anaphase transition. While much of USP16 is localized in the cytoplasm, separating the enzyme from its substrates is considered an important regulatory mechanism. Some of the functions that USP16 has been linked to include DNA damage repair, immune disease, tumorigenesis, protein synthesis, coronary artery health, and male infertility. The strong connection to immune response and the fact that multiple oncogene products are substrates of USP16 suggests that USP16 may be a potential therapeutic target for the treatment of certain human diseases.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.

BACKGROUND: Gout is a common and complex form of immunoreactive arthritis based on hyperuricemia, while the symptoms would turn to remission or even got worse. So, it is hard to early identify whether an asymptomatic hyperuricemia (AHU) patient will be susceptible to get acute gout attack and it is also hard to predict the process of gout remission to flare. Here, we report that the plasma proteins profile can distinguish among acute gout (AG), remission of gout (RG), AHU patients, and healthy controls.
METHODS: We established an isobaric tags for relative and absolute quantification (iTRAQ) and parallel reaction monitoring (PRM) based method to measure the plasma proteins for AG group (n = 8), RG group (n = 7), AHU group (n = 7) and healthy controls (n = 8).
RESULTS: Eleven differentially expressed proteins such as Histone H2A, Histone H2B, Thrombospondin-1 (THBS1), Myeloperoxidase (MPO), Complement C2, Complement component C8 beta chain (C8B), Alpha-1-acid glycoprotein 1 (ORM1), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Carbonic anhydrase 1 (CA1), Serum albumin (ALB) and Multimerin-1 (MMRN1) were identified. Histone H2A, Histone H2B and THBS1 might be the strongest influential regulator to maintain the balance and stability of the gout process. The complement and coagulation cascades is one of the main functional pathways in the mechanism of gout process.
CONCLUSIONS: Histone H2A, Histone H2B and THBS1 are potential candidate genes for novel biomarkers in discriminating gout attack from AHU or RG, providing new theoretical insights for the prognosis, treatment, and management of gout process.
BACKGROUND: This study is not a clinical trial.

The E3 ubiquitin ligase RAD18 mono-ubiquitinates PCNA to promote bypass of replication fork-stalling DNA lesions. On the other hand, RAD18 also contributes to DNA double-strand break (DSB) repair. RAD18 is recruited to ionizing radiation (IR)-induced DSB and colocalizes with ubiquitinated chromatin proteins. RAD18 interacts with the ubiquitinated chromatin proteins via its ubiquitin-binding Zinc finger (UBZ) domain and is proposed to propagate DNA DSB signalling and recruit DNA repair proteins. We found that purified human RAD18 protein complexed with RAD6B (RAD6B-RAD18) catalyzes mono- and poly-ubiquitination of histone H2A in vitro while UBZ domain-mutated RAD18 complexed with RAD6B protein catalyzes mono- but not poly-ubiquitination of histone H2A. Human RAD18-/-cells synchronized at the G1 phase show a reduced signal of ubiquitinated protein in chromatin after IR when compared to that of wild-type control cells. The reduced signal of ubiquitinated protein in RAD18-/-cells is rescued by the introduction of RAD18 cDNA but to a lesser extent by the introduction of cDNA coding RAD18 lacking UBZ domain. Taken together, these results indicate that RAD18 mediates DSB-induced ubiquitination of chromatin protein during the G1 phase.

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.

Timely completion of DNA replication is central to accurate cell division and to the maintenance of genomic stability. However, certain DNA-protein interactions can physically impede DNA replication fork progression. Cells remove or bypass these physical impediments by different mechanisms to preserve DNA macromolecule integrity and genome stability. In Saccharomyces cerevisiae, Wss1, the DNA-protein crosslink repair protease, allows cells to tolerate hydroxyurea-induced replication stress, but the underlying mechanism by which Wss1 promotes this function has remained unknown. Here, we report that Wss1 provides cells tolerance to replication stress by directly degrading core histone subunits that non-specifically and non-covalently bind to single-stranded DNA. Unlike Wss1-dependent proteolysis of covalent DNA-protein crosslinks, proteolysis of histones does not require Cdc48 nor SUMO-binding activities. Wss1 thus acts as a multi-functional protease capable of targeting a broad range of covalent and non-covalent DNA-binding proteins to preserve genome stability during adverse conditions.

Background: Ras-PI3K pathway aberrant activation plays an important role in the occurrence and development of osteosarcoma. This study investigated the functions of Ras-PI3K pathway specific activation on histone H2A phosphorylation at threonine 120 (H2AT120ph) in osteosarcoma cells, along with the possible internal molecular mechanisms.Methods: Cell transfection was done to alter RasG12V/Y40C, H2AT120ph and vaccinia-related kinase 1 (VRK1) expression. Then, cell viability, proliferation, migration and cell cycle distribution were assessed, respectively. qRT-PCR was utilized to measure the VRK1 and Ras-PI3K pathway downstream genes (CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16) expression. Chromatin immunoprecipitation (ChIP) was conducted to evaluate the input levels of H2AT120ph and VRK1 in the promoter regions of Ras-PI3K pathway downstream genes.Results: Ras-PI3K specific activation promoted histone H2AT120ph. H2AT120ph participated in the oncogenic functions of Ras-PI3K pathway on osteosarcoma by modulating the transcription of Ras-PI3K-targeted genes. Moreover, VRK1 contributed to the Ras-PI3K specific activation-induced up-regulation of H2AT120ph and osteosarcoma progression. Ras-PI3K pathway-specific activation-induced up-regulation of H2AT120ph was achieved by up-regulation of VRK1.Conclusions: Ras-PI3K pathway activation promoted osteosarcoma progression might be via up-regulating VRK1-mediated H2AT120ph. We proposed that VRK1 and H2AT120ph could be the potential targets for osteosarcoma diagnosis and treatment.HighlightsH2AT120ph is specifically promoted by Ras-PI3K pathway activation.H2AT120ph joins in the oncogenic effects of Ras-PI3K pathway on osteosarcoma.H2AT120ph regulates the transcription of Ras-PI3K-targeted genes.VRK1 takes part in the regulatory function of Ras-PI3K pathway on H2AT120ph.

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.

DNA topoisomerases are considered consolidated druggable targets against diseases produced by trypanosomatids. Several reports indicated that indenoisoquinolines, a family of non-camptothecinic based topoisomerase poisons, have a strong leishmanicidal effect both in vitro and in vivo in murine models of visceral leishmaniasis. The antileishmanial effect of the indenoisoquinolines implies several mechanisms that include the stabilization of the cleavage complex, histone H2A phosphorylation and DNA fragmentation. A series of 20 compounds with the indenoisoquinoline scaffold and several substituents at positions N6, C3, C8 and C9, were tested both in promastigotes and in intramacrophage splenic amastigotes obtained from an experimental murine infection. The antileishmanial effect of most of these compounds was within the micromolar or submicromolar range. In addition, the introduction of an N atom in the indenoisoquinoline ring (7-azaindenoisoquinolines) produced the highest selectivity index along with strong DNA topoisomerase IB inhibition, histone H2A phosphorylation and DNA-topoisomerase IB complex stabilization. This report shows for the first time the effect of a series of synthetic indenoisoquinolines on histone H2A phosphorylation, which represents a primary signal of double stranded DNA break in genus Leishmania.