Expression Of Histone H3 Research Articles

Occlusal disharmony promotes anxiety-like behaviours by suppressing Sirt1.

Previous studies have indicated that occlusal disharmony (OD) can promote anxiety-like behaviours. However, the specific molecules involved in the development of anxiety-like behaviours and their underlying mechanisms remain unknown. OD was produced by anterior crossbite of female mice. We measured the anxiety levels of mice in each group and screened the hippocampal mRNA expression profiles of mice in the control group and OD group. The role of target mRNA in OD-induced anxiety-like behaviours was evaluated and we preliminarily explored the possible downstream pathways. The results suggested that OD can induce and promote anxiety-like behaviours with/without chronic unpredictable mild stress. We found that Sirt1 was significantly downregulated within the hippocampus in OD mice. In addition, the downregulation of Sirt1 within the hippocampus in OD and control mice promoted anxiety-like behaviours, increased acetylated histone H3 expression and decreased Dnah12 transcription levels. In contrast, in OD mice subjected to an injection of resveratrol, there was a remission of anxiety-like behaviours and an upregulation of Sirt1 in the hippocampus, the effects of which were accompanied by decreased acetylated histone H3 expression and increased Dnah12 transcription levels. OD leads to increased sensitivity to chronic stress in mice, resulting in anxiety-like behaviours. During this process, Sirt1 acts as an effective factor in the regulation of OD-induced anxiety-like behaviours. OD, as a stressor, could induce anxiety-like behaviours. It investigates the impact of OD (a stressor) on the molecular genetic of the pathophysiology of major neuropsychiatric disorders.

The NRF2-CARM1 axis links glucose sensing to transcriptional and epigenetic regulation of the pentose phosphate pathway in gastric cancer

Cancer cells autonomously alter metabolic pathways in response to dynamic nutrient conditions in the microenvironment to maintain cell survival and proliferation. A better understanding of these adaptive alterations may reveal the vulnerabilities of cancer cells. Here, we demonstrate that coactivator-associated arginine methyltransferase 1 (CARM1) is frequently overexpressed in gastric cancer and predicts poor prognosis of patients with this cancer. Gastric cancer cells sense a reduced extracellular glucose content, leading to activation of nuclear factor erythroid 2-related factor 2 (NRF2). Subsequently, NRF2 mediates the classic antioxidant pathway to eliminate the accumulation of reactive oxygen species induced by low glucose. We found that NRF2 binds to the CARM1 promoter, upregulating its expression and triggering CARM1-mediated hypermethylation of histone H3 methylated at R arginine 17 (H3R17me2) in the glucose-6-phosphate dehydrogenase gene body. The upregulation of this dehydrogenase, driven by the H3R17me2 modification, redirects glucose carbon flux toward the pentose phosphate pathway. This redirection contributes to nucleotide synthesis (yielding nucleotide precursors, such as ribose-5-phosphate) and redox homeostasis and ultimately facilitates cancer cell survival and growth. NRF2 or CARM1 knockdown results in decreased H3R17me2a accompanied by the reduction of glucose-6-phosphate dehydrogenase under low glucose conditions. Collectively, this study reveals a significant role of CARM1 in regulating the tumor metabolic switch and identifies CARM1 as a potential therapeutic target for gastric cancer treatment.

Mck1-mediated proteolysis of CENP-A prevents mislocalization of CENP-A for chromosomal stability in Saccharomyces cerevisiae.

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1-mediated phosphorylation of Skp, Cullin, F-box-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL-cse4-3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

Lysine Methylation and Histone Modifications during Cold Stress of Insects: Freeze-Tolerant Eurosta solidaginis and Freeze-Avoiding Epiblema scudderiana.

Overwintering survival by insects, whether of the freeze-tolerant or freeze-avoiding types, is typically associated with a strong suppression of metabolic rate (e.g., entry into diapause) that involves the differential expression of many genes with regulation at the transcriptional, translational or post-translational levels. Epigenetic modifications have been suggested to play a vital role in regulating cold responses of insects. However, knowledge of the roles of epigenetic mechanisms in modulating gene expression for winter survival of the larvae of two goldenrod gall formers, the freeze-tolerant dipteran Eurosta solidaginis and the freeze-avoiding lepidopteran Epiblema scudderiana, remain unknown. The current study evaluates the role of cold-induced lysine methylation and histone modifications, with enzymes of lysine methylation (SETD8, SETD7, SUV39H1, SMYD2 and ASH2L), as well as relative levels of histone H3 acetylation (H3K9ac, H3K18ac, H3K27ac, H3K56ac) and methylation (H3K4me1, H3K9me3, H3K36me2) examined in two insects. Significant (p < 0.05) reductions were observed in most of the targets of histone methylation/acetylation for decreasing temperatures of Ep. scudderiana larvae, whereas selected histone methylation/acetylation targets were conversely elevated (p < 0.05) in E. solidaginis, particularly under conditions of 5 °C for 4 h. Histone H3 expression was found to be variable without statistical differences in larval goldenrod gall moths and gall flies. These results provide basic information on the patterns of epigenetic regulation involved in insect cold hardiness.

Calcineurin/NFATc1 pathway represses cellular cytotoxicity by modulating histone H3 expression

Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.

Exposure to nickel chloride induces epigenetic modification on detoxification enzyme glutathione S-transferase M2.

Nickel (Ni) is a human carcinogen with genotoxic and epigenotoxic effects. Environmental and occupational exposure to Ni increases the risk of cancer and chronic inflammatory diseases. Our previous findings indicate that Ni alters gene expression through epigenetic regulation, specifically impacting E-cadherin and angiopoietin-like 4 (ANGPTL4), involved in epithelial-mesenchymal transition and migration. GST-M2, a member of the glutathione S-transferase (GST) enzyme family, plays a crucial role in cellular defense against oxidative damage and has been increasingly associated with cancer. GST-M2 overexpression inhibits lung cancer invasion and metastasis in vitro and in vivo. Hypermethylation of its promoter in cancer cells reduces gene expression, correlating with poor prognosis in non-small-cell lung cancer patients. The impact of Ni on GST-M2 remains unclear. We will investigate whether nickel exerts regulatory effects on GST-M2 through epigenetic modifications. Additionally, metformin, an antidiabetic drug, is being studied as a chemopreventive agent against nickel-induced damage. Our findings indicate that nickel chloride (NiCl2 ) exposure, both short-term and long-term, represses GST-M2 expression. However, the expression can be restored by demethylation agent 5-aza-2'-deoxycytidine and metformin. NiCl2 promotes hypermethylation of the GST-M2 promoter, as confirmed by methylation-specific PCR and bisulfite sequencing. Additionally, NiCl2 also influences histone acetylation, and metformin counteracts the suppressive effect of NiCl2 on histone H3 expression. Metformin reestablishes the binding of specificity protein 1 to the GST-M2 promoter, which is otherwise disrupted by NiCl2 . These findings elucidate the mechanism by which Ni reduces GST-M2 expression and transcriptional activity, potentially contributing to Ni-induced lung carcinogenesis.

Effect of Lysine Acridone Acetate on the Level of Apoptosis and Expression of Apoptosis-Associated Proteins during Antioncogenic Therapy in Colorectal Cancer in Mice.

We studied the mechanism of action of cytostatics with the addition of lysine acridone acetate to evaluate the possibility of its use for improving the effectiveness of antioncogenic therapy in colorectal cancer. In Nude mouse model, the level of apoptosis (TUNEL) and expression of proteins CD95, p53, Bcl-2, histone H3, and Ki-67 (immunohistochemistry) were assessed in primary tumor biopsy specimens. It has been shown that cytostatic treatment led to stimulation of p53-mediated apoptosis and suppression of proliferation (Ki-67 expression) of tumor cells, and apoptosis level was increased in groups receiving lysine acridone acetate. H3 expression in the experimental groups was changed.

Interactions between Integrin α9β1 and VCAM-1 Promote Neutrophil Hyperactivation and Deep Vein Thrombosis in a Murine Model of Lung Cancer

Background: Venous thromboembolism (VTE) is highly prevalent in patients with lung cancer and is associated with increased morbidity and mortality. Ectopic secretion of granulocyte colony-stimulating factor (G-CSF), a critical regulator of neutrophil trafficking, is commonly observed in lung cancer. In recent years, compelling evidence has emerged implicating neutrophils and neutrophil extracellular traps (NETosis) in cancer-associated thrombosis. Integrin α9β1 is highly expressed on neutrophils, upregulated upon neutrophil activation, promotes NETosis, and is known to stabilize neutrophil adhesion to activated endothelium via multiple ligands including vascular cell adhesion molecule 1 (VCAM-1). Interactions between integrin α9 and VCAM-1 have been reported to reduce neutrophils apoptosis. Given the relevance of integrin α9 in neutrophil migration and apoptosis, granulopoiesis, and thromboinflammation, here we evaluated the mechanistic role of neutrophil integrin α9β1 in the pathophysiology of cancer-associated thrombosis. Methods: Lung cancer was induced by the intravenous administration of 3X10 5 Lewis lung carcinoma cells. Deep vein thrombosis (DVT) was induced in neutrophil-specific α9 -/- (α9 fl/flMrp8Cre +/-) and littermate controls (α9 fl/flMrp8Cre -/-) by partial ligation of the inferior vena cava (IVC). Plasma levels of the thrombin-antithrombin (TAT) complex, fibrinogen, G-CSF, elastase, and myeloperoxidase (MPO) were assessed by ELISA. To evaluate the potential mechanisms by which neutrophil integrin α9-VCAM-1 interactions promote DVT, we performed unbiased RNA sequencing from 1) control neutrophils stimulated with and without VCAM-1 and, 2) neutrophils of α9 fl/flMrp8Cre +/- and α9 fl/flMrp8Cre -/- mice stimulated with VCAM-1. Computational modeling, docking, virtual screening, and the cell-free assay were used to identify small-molecule inhibitors of integrin α9-VCAM-1 interaction. Results: Mice with lung cancer exhibited significantly increased levels of TAT, G-CSF, blood neutrophil/lymphocyte ratio, cell-free DNA, elastase, and MPO, suggesting a hypercoagulable and inflammatory state. Tumor-bearing and non-tumor-bearing neutrophil-specific α9 -/- mice exhibited reduced DVT severity, as evident by reduced IVC thrombus weight and thrombus length compared to littermate controls, while thrombosis incidence was comparable between the groups. Reduced DVT severity in neutrophil-specific α9 -/- mice was also associated with decreased neutrophil content and citrullinated histone H3 (CitH3) expression in IVC thrombi. Integrin α9 deficient neutrophils incubated with VCAM-1 exhibited reduced release of elastase and MPO, decreased endothelial cell apoptosis, and reduced adhesion to the activated endothelium at venous shear rate compared to control neutrophils incubated with VCAM-1. Bioinformatic analysis of RNA sequencing data from VCAM-1 stimulated neutrophils from α9 fl/flMrp8Cre +/- and control mice showed distinct transcriptional profiles. Hierarchical clustering of gene expression revealed gene clusters that were highly downregulated in stimulated neutrophils from α9 fl/flMrp8Cre +/- mice. Gene ontology enrichment revealed that highly downregulated genes corresponded to biological processes involved in the inflammatory response, cytokine production, NF-κB and ERK signaling pathways, granulocyte migration, and chemotaxis. Virtual screening and cell-free assay identified macitentan as a potent inhibitor of integrin α9-VCAM-1 interactions. In vitro treatment with macitentan reduced neutrophil adhesion to VCAM-1 and also to activated endothelial cells at venous shear rate. In vivo, macitentan treatment at a dose of 5 mg/kg exhibited reduced DVT severity, decreased plasma levels of elastase and MPO, and reduced neutrophil and CitH3 levels in IVC thrombi. Conclusion: Using genetic and pharmacological approaches in combination with transcriptomic and pharmacological studies, we described a previously unknown pathway involving integrin α9 and VCAM-1 in promoting cancer-associated thrombosis. We found that integrin α9-VCAM1 axis is critical for neutrophil hyperactivation, and its pharmacological inhibition significantly reduces DVT severity. The results of this study suggest that neutrophil integrin α9 is a key factor regulating neutrophil hyperactivation and cancer-associated thrombosis.

Abstract 17359: UCP2 Induced Glycolysis Promotes Histone Acetylation to Regulate Cardiomyocyte Cell Cycle After Injury

Developmental cardiac tissue is proliferative capable of robust regenerative response after myocardial injury, but this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Postnatal CMs exhibit a specialized metabolic state that supports rapid proliferation and is altered with loss of regeneration in the heart. Studies have shown that reliance on glycolysis for energy generation is preferred by proliferating CMs and activation of glycolysis promotes cardiac repair. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle and repair in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM cell cycle dynamics. UCP2 was found to be primarily active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression in human IPS derived CMs increases cell cycle progression as assessed by immunostaining and flow cytometry-based DNA content analysis. In parallel, neonatal rat ventricular myocytes (NRVMs) overexpressing UCP2 showed increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. Next, we generated αMHC-UCPKO mice that showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, UCP2 induced glycolysis increases ATP-citrate lyase (ACLY) mediated acetyl-CoA generation in the CMs. Additionally, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.

The Role of Acetylation and Methylation of Rat Hippocampal Histone H3 in the Mechanism of Aluminum-Induced Neurotoxicity.

Aluminum is a known neurotoxin and a major environmental contributor to neurodegenerative diseases such as Alzheimer's disease (AD). We uesd a subchronic aluminum chloride exposure model in offspring rats by continuously treating them with AlCl3 solution from the date of birth until day 90 in this research. Then evaluated the neurobehavioral changes in rats, observed the ultrastructural changes of hippocampal synapses and neurons, and examined the level of hippocampal acetylated histone H3 (H3ac), the activity and protein expression of hippocampal HAT1 and G9a, and the protein expression level of H3K9 dimethylation (H3K9me2). The findings demonstrated that aluminum-treated offspring rats had impaired learning and memory abilities as well as ultrastructural alterations in hippocampal synapses and neurons. The level of histone H3ac was decreased along with decreased protein expression and activity of HAT1, while level of H3K9me2 was increased along with increased protein expression and activity of G9a.

Apoptotic bodies of bone marrow mesenchymal stem cells inhibit endometrial stromal cell fibrosis by mediating the Wnt/β-catenin signaling pathway

BackgroundIntrauterine adhesions (IUAs) are a common illness of the uterine cavity. Endometrial fibrosis is the main pathological feature. In addition to a high recurrence rate, patients with severe IUAs have a low pregnancy rate. However, there are few effective treatments for IUAs. This study aims to confirm the influence of apoptotic bodies of bone marrow mesenchymal stem cells (BMSCs) on endometrial stromal cell fibrosis by mediating the Wnt/β-catenin signaling pathway and to provide new insight for the clinical treatment of IUAs. MethodsHuman endometrial stromal cells (HESCs) were used to establish an IUA cell model by treatment with TGF-β1, and a rat IUA model was established by the double injury method. Apoptosis of BMSCs was detected by TUNEL assays, and cell morphology was observed by the CM-DiI tracer. The morphology of apoptotic vacuoles and apoptotic bodies (ABs) was detected by TEM. We used Western blotting to detect the expression of histone H3.3, histone H2B, C3b, cyclin D1, C1QC, α-SMA, COL1A1, COL5A2, FN, CTGF, Wnt2b, c-MYC, CK-18 and VIM. The expression levels of α-SMA, COL1A1, COL5A2, FN and CTGF were detected by RT‒qPCR. The expression levels of α-SMA, COL1A1, FN and CTGF were detected by immunofluorescence. Immunohistochemistry was used to detect the expression of TGF-β, CK-18 and VIM. Flow cytometry, cell scratch assays, CCK-8 assays, and H & E and Masson staining were used to detect the cell cycle, cell migration, cell proliferation, and endometrial pathology, respectively. ResultsWe found that ultraviolet light (UV) irradiation induced apoptosis of BMSCs and increased the production of ABs. TGF-β1 treatment can induce HESCs to form extracellular matrix (ECM), and aggravate cell fibrosis, and adding ABs or FH535, an inhibitor of the Wnt/β-catenin signaling pathway, can inhibit TGF-β1-induced HESC fibrosis. However, the inhibitory effect of ABs on TGF-β1-induced fibrosis of HESCs was attenuated by the addition of LiCl. In the Wnt/β-catenin signaling pathway, LiCl is an activator after coculture with TGF-β1. In vivo, IUA-induced narrowing of the uterine cavity was accompanied by intrauterine adhesions, increased deposition of collagen fibers, upregulation of TGF-β1, VIM, α-SMA, COL1A1 and COL5A2, and downregulation of CK-18. These changes in expression were reversed after treatment with ABs or FH535. When ABs and LiCl were added at the same time, the inhibitory effect of ABs on IUA fibrosis was weakened. ConclusionBMSC-derived ABs inhibit the fibrosis of HESCs by inhibiting the Wnt/β-catenin signaling pathway. These results provide a new direction for the clinical treatment of IUAs.

Significance of Histone H3.3 (G34W)-Mutant Protein in Pathological Diagnosis of Giant Cell Tumor of Bone.

This study aimed to examine the expression of Histone H3.3 glycine 34 to tryptophan (G34W) mutant protein in Giant Cell Tumor of Bone (GCTB). This analytic observation research used a cross-sectional study design on 71 bone tumors. The cases involved 54 tissue samples diagnosed as GCBT. It was divided into GCTB primer (n=37), recurrent GCTB (n=5), GCTB with metastasis (n=9), and malignant GCTB (n=3). There were 17 samples mimics of GCTB also tested, including chondroblastoma (n=1), giant cell reparative granuloma (n=2), giant cell of tendon sheath (n=7), chondromyxoid fibroma (n=2), aneurysmal bone cyst (n=2), and giant cell-rich osteosarcoma (n=3). The Immunohistochemistry was used to evaluate the expression of G34W-mutated protein in these bone tumors. The representation H3.3 (G34W) was expressed in the nuclei of mononuclear stromal cells but not stained on osteoclast-like giant cells. This study was analyzed by the Chi-square test, Fisher's test, specificity test, and sensitivity test. We obtained p = 0.001 for Histone H3.3 (G34W) mutant expression in GCTB vs Non-GCTB. Statistically, there was no significant difference in the expression level of Histone H3.3 (G34W) in the GCTB and its variants p-value = 0.183. We also obtained that the specificity of Histone H3.3 expression on GCTB was 100% and the sensitivity of Histone H3.3 on GCTB was 77.8%. Histon H3.3 mutant as a mutated driver gene in an Indonesian GCTB can assist to diagnose GCTB and compare it from other bone tumors.

Tributyrin Intake Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm in LDLR-/- Mice.

Abdominal aortic aneurysm (AAA) is a multifactorial cardiovascular disease with a high risk of death, and it occurs in the infrarenal aorta with vascular dilatation. High blood pressure acts on the aortic wall, resulting in rupture and causing life-threatening intra-abdominal hemorrhage. Vascular smooth muscle cell (VSMC) dysregulation and extracellular matrix (ECM) degradation, especially elastin breaks, contribute to structural changes in the aortic wall. The pathogenesis of AAA includes the occurrence of oxidative stress, inflammatory cell infiltration, elastic fiber fragmentation, VSMC apoptosis, and phenotypic transformation. Tributyrin (TB) is decomposed by intestinal lipase and has a function similar to that of butyrate. Whether TB has a protective effect against AAA remains uncertain. In the present study, we established an AAA murine model by angiotensin II (AngII) induction in low-density lipoprotein receptor knockout (LDLR-/-) mice and investigated the effects of orally administered TB on the AAA size, ratio of macrophage infiltration, levels of matrix metalloproteinase (MMP) expression, and epigenetic regulation. TB attenuates AngII-induced AAA size and decreases elastin fragmentation, macrophage infiltration, and MMP expression in the medial layer of the aorta and reduces the levels of SBP (systolic blood pressure, p < 0.001) and MMP-2 (p < 0.02) in the serum. TB reduces the AngII-stimulated expression levels of MMP2 (p < 0.05), MMP9 (p < 0.05), MMP12, and MMP14 in human aortic smooth muscle cells (HASMCs). Moreover, TB and valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, suppress AngII receptor type 1 (AT1R, p < 0.05) activation and increase the expression of acetyl histone H3 by HDAC activity inhibition (p < 0.05). Our findings suggest that TB exerts its protective effect by suppressing the activation of HDAC to attenuate the AngII-induced AT1R signaling cascade.

Developmentally programmed histone H3 expression regulates cellular plasticity at the parental-to-early embryo transition.

During metazoan development, the marked change in developmental potential from the parental germline to the embryo raises an important question regarding how the next life cycle is reset. As the basic unit of chromatin, histones are essential for regulating chromatin structure and function and, accordingly, transcription. However, the genome-wide dynamics of the canonical, replication-coupled (RC) histones during gametogenesis and embryogenesis remain unknown. In this study, we use CRISPR-Cas9-mediated gene editing in Caenorhabditis elegans to investigate the expression pattern and role of individual RC histone H3 genes and compare them to the histone variant, H3.3. We report a tightly regulated epigenome landscape change from the germline to embryos that are regulated through differential expression of distinct histone gene clusters. Together, this study reveals that a change from a H3.3- to H3-enriched epigenome during embryogenesis restricts developmental plasticity and uncovers distinct roles for individual H3 genes in regulating germline chromatin.

Abstract 2021: Naveni TriFlex cell: An emerging method for simultaneous detection of free proteins and their interactions

Abstract Determining the levels of protein-protein interactions (PPI) is essential for the understanding of signal transduction, regulation of gene expression and mutation effects, infection mechanisms, identification of diagnostic markers, etc. Taking the already challenging task of PPI identification a step further, the next aim is to determine the levels of non-interacting proteins in parallel. The ability to concurrently monitor free and interacting proteins opens the door to studying their functional states and interplay, thus gaining deeper insight from a single staining experiment. Additionally, to accomplish this in situ, retaining the structural integrity of the cell, helps understand spatiotemporal communication between proteins in their native environment. To this end, we designed Naveni TriFlex Cell – a highly sensitive and specific proximity-based technology, relying on two user-determined primary antibodies against the targets of interest, and on proprietary TriFlex Navenibodies, which are antibody-based proximity reagents. TriFlex Cell detects total protein A (i.e., both free and in complex with B), total protein B, and the AB interaction. The detected A, B and AB signals are amplified and generate fluorescent readout in three channels corresponding to each protein pool. As proof of principle, we stained MCF7 cells for E-cadherin and β-catenin, which are known to interact in the adherens junctions. We observed highly abundant interactions at the cell membrane, some complexes and free proteins in the cytoplasm, and very low background in controls where either or both primary antibodies were omitted. These results attested to the high sensitivity and specificity of our method. Next, we explored the expression and interaction of Histone H3 and Lamin B, a nuclear envelope protein. Despite the compact structure of the nucleus, we were able to distinguish individual signals and abundant interactions, with free Lamin B particularly enriched in the nuclear membrane. In dividing cells, we observed diffuse Lamin signals in line with nuclear envelope breakdown, and more densely packed Histone H3 associated with chromatin. In contrast to non-mitotic cells, interactions were few, but increasing with the advance from metaphase to telophase. Finally, we stained against GM130, a Golgi complex protein, and COX1, a mitochondrial marker, as a biological non-interaction control experiment. TriFlex Cell successfully detected the individual proteins in their respective subcellular locations, and no complexes. In summary, our data demonstrate that Naveni TriFlex Cell is a sensitive and specific method that visualizes low and high abundant targets in various cell compartments and adds value by uncovering protein interplay, such as complex formation/dissolution under dynamic biological conditions. Citation Format: Axel Klaesson, Doroteya Raykova, Agata Zieba Wicher. Naveni TriFlex cell: An emerging method for simultaneous detection of free proteins and their interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2021.

Upregulation of histone H3 caused by CRYAA may contribute to the development of age-related cataract

Objective: Age-relate cataract (ARC) is a disease of the eyes with no effective drugs to prevent or treat patients. The aim of the present study is to determine whether histone H3, αA-crystallin (CRYAA), β-galactosidase (GLB1), and p53 are involved in the pathogenesis of ARC. Methods: A total of 99 anterior lens capsules (ALCs) of patients with ARC of various nuclear grades, ultraviolet models of ALCs, and two human lens epithelial cell lines (FHL-124 and SRA01/04) were used, and the expression of histone H3, CRYAA, GLB1, and p53 were detected by immunoblotting and reverse transcription and real time-quantitative polymerase chain reaction. The association between CRYAA with histone H3, GLB1, and p53 was assessed in FHL-124 and SRA01/04 cells following CRYAA overexpression. Results: Histone H3 and p53 in ALCs of patients with ARC were up-regulated in a grade-dependent manner, and the expression of CRYAA showed a positive association with histone H3, p53, and GLB1. In UV models of ALCs and human lens epithelial cell lines, the expression levels of histone H3, cell apoptosis factors (Bax/Bcl-2, cleaved caspase-3), and inflammation factors (interleukin-6, tumor necrosis factor-α) were all up-regulated. Furthermore, transfection of CRYAA in FHL-124 cells induced overexpression of histone H3. Conclusion: CRYAA-mediated upregulation of histone H3 may be involved in the pathogenesis of ARC. p53 may also have a role in ARC development, but not via the CRYAA-histone H3 axis. The results of the present study may assist in improving our understanding of the pathogenesis of ARC and in identifying potential targets for treatment.

Extracellular histone H3 facilitates ferroptosis in sepsis through ROS/JNK pathway.

Previous evidence realized the critical role of histone in disease control. The anti-inflammatory function of estradiol (E2) in sepsis has been documented. We here intended to unveil the role of extracellular histone H3 in sepsis regarding cell ferroptosis and the role of E2 in a such mechanism. Clinical sample, cecal ligation and puncture (CLP)-induced animal models and lipopolysaccharides (LPS)-induced cell models were prepared for testing relative expression of extracellular histone H3 and E2 as well as analyzing the role of extracellular histone H3 and E2 in sepsis concerning cell viability, reactive oxygen species (ROS), and ferroptosis. Under sepsis, we found increased ferroptosis and extracellular histone H3 content, but reduced E2 concentration. Extracellular histone H3 facilitated ferroptosis of human umbilical vein endothelial cells(HUVECs) induced by LPS through activating the ROS/c-Jun N-terminal kinase (JNK)pathway. Moreover, E2 antagonized the effect of extracellular histone H3 on LPS-induced HUVEC ferroptosis and sepsis injury in CLP-induced animal models. We highlighted that extracellular histone H3 facilitated lipopolysaccharides-induced HUVEC ferroptosis via activating ROS/JNK pathway, and such an effect could be antagonized by E2.

Epigenetic modification of Kiss1 gene expression in the AVPV is essential for female reproductive aging.

Female reproductive senescence is heralded by hypothalamus region-specific changes in the transcription of genes such as Kiss1 under estradiol (E2) positive feedback, associated with luteinizing hormone (LH) surge dysfunction and reproductive decline. The current study explored whether the anteroventral periventricular nucleus (AVPV) displayed epigenetic changes mediated by age-related dysregulation of gene expression and whether an epigenetic-based intervention could alleviate an aging-related neuroendocrine disorder. Chromatin immunoprecipitation sequencing (ChIP-seq) and ChIP-qPCR were used to assess the differential acetylation of histone H3 in the AVPV and the expression of genes in hormone-primed middle-aged rats. The association between acetylated histone H3 and Kiss1 expression and the underlying mechanisms of dysregulation were determined using pharmacological inhibitors and molecular experiments in vitro and in vivo. An AVPV gene expression program failed to initiate in middle-aged females displaying typical genome-wide hypoacetylation of histone H3, and this coincided with decreased LH. Hypoacetylation of histone H3 at the 3' intergenic region of Kiss1 in particular was associated with enhanced chromatin looping between the promoter and enhancer. Restoration of physiological histone H3 acetylation by intracerebroventricular injection of trichostatin A (TSA) restored the expression of Kiss1 by modifying chromatin looping and led to the restoration of Kiss1 neuronal activation and Kiss1 synthesis as well as circulating LH. These findings have revealed novel epigenetic-associated changes in gene expression in female reproductive aging. These results also suggest that HDAC enzyme-based treatment is a potential therapeutic approach for insufficient preovulatory LH release in aging females.

An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation

BackgroundOwing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PurposeTo obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. MethodsDiethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. ResultsFrom HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-β-Manp-(1→, β-Manp-(1→ and →3)-β-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 μg/ml and 307 ± 11 μg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. ConclusionAn anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.

Abstract P2007: Bmal1 Drives Postnatal Cardiac Hypertrophy Via Circadian Chromatin Remodeling Of The Pro-hypertrophic Gene Sik1

Background: During neonatal cardiac development, myocytes integrate cues such as changes in blood pressure and circulating hormones to execute a temporal genetic response that drives physiological cardiac hypertrophy. Chromatin remodeling precedes the adult heart’s response to growth stimuli, yet the temporal nature of neonatal chromatin remodeling is unclear. In other tissues, genes regulated by the master circadian rhythm factor BMAL1 display circadian oscillations in histone acetylation and nucleosome and RNAP II occupancy. We hypothesized that BMAL1 regulates temporal chromatin remodeling and expression of genes critical for neonatal cardiac hypertrophy. Methods: BMAL1 chromatin immunoprecipitation sequencing (ChIP-Seq) and assay for transposase-accessible chromatin and sequencing (ATAC-Seq) data from murine hearts were analyzed to identify genes at which BMAL1 may drive chromatin remodeling. To interrogate myocyte time-of-day dependent response to growth stimuli, neonatal rat ventricular myocytes (NRVM) were subjected to serum shock synchronization to drive oscillatory BMAL1 expression, followed by treatment with the growth stimulus phenylephrine (PE) at the peak and trough of BMAL1 expression. BMAL1 was knocked down via siRNA and hypertrophy was assessed by measurement of cell size and of RT-PCR of fetal genes (Nppa and Nppb). Results: BMAL1 ChIP-Seq data revealed myocyte-specific BMAL1 localization to regions exhibiting an open chromatin signature based on ATAC-seq, including the pro-hypertrophic gene salt-inducible kinase 1 ( Sik1 ). Serum shock induced oscillations of BMAL1 and histone H3.3: administration of PE at the peak of BMAL1 expression induced a 30% increase in cell size and 2-fold increase in fetal gene expression. PE given at the trough of BMAL1 expression affected neither cell size nor fetal genes, demonstrating a critical role for the circadian clock in myocyte growth. BMAL1 knockdown: decreased expression of its known target and partner clock gene, Per2 , as well as expression of Sik1, Nppa , and Nppb ; decreased myocyte size by 30%; and increased expression of histone H3.3. Conclusions: These data indicate that BMAL1 temporally regulates myocyte growth by modulating histone stoichiometry and expression of Sik1 .