Histone Research Articles

Design, Synthesis, and Biological Evaluation of HDAC Inhibitors Containing Natural Product-Inspired N-Linked 2-Acetylpyrrole Cap.

Drawing inspiration from the structural resemblance between a natural product N-(3-carboxypropyl)-2-acetylpyrrole and phenylbutyric acid, a pioneer HDAC inhibitor evaluated in clinical trials, we embarked on the design and synthesis of a novel array of HDAC inhibitors containing an N-linked 2-acetylpyrrole cap by utilizing the pharmacophore fusion strategy. Among them, compound 20 exhibited potential inhibitory activity on HDAC1, and demonstrated notable potency against RPMI-8226 cells with an IC50 value of 2.89 ± 0.43 μM, which was better than chidamide (IC50 = 10.23 ± 1.02 μM). Western blot analysis and Annexin V-FTIC/propidium iodide (PI) staining showed that 20 could enhance the acetylation of histone H3, as well as remarkably induce apoptosis of RPMI-8226 cancer cells. The docking study highlighted the presence of a hydrogen bond between the carbonyl oxygen of the 2-acetylpyrrole cap group and Phe198 of the HDAC1 enzyme in 20, emphasizing the crucial role of introducing this natural product-inspired cap group. Molecular dynamics simulations showed that the docked complex had good conformational stability. The ADME parameters calculation showed that 20 possesses remarkable theoretical drug-likeness properties. Taken together, these results suggested that 20 is worthy of further exploration as a potential HDAC-targeted anticancer drug candidate.

Mitotic chromatin marking governs asymmetric segregation of DNA damage.

The faithful segregation of intact genetic material and the perpetuation of chromatin states through mitotic cell divisions are pivotal for maintaining cell function and identity across cell generations. However, most exogenous mutagens generate long-lasting DNA lesions that are segregated during mitosis. How this segregation is controlled is unknown. Here, we uncover a mitotic chromatin-marking pathway that governs the segregation of UV-induced damage in human cells. Our mechanistic analyses reveal two layers of control: histone ADP-ribosylation, and the incorporation of newly synthesized histones at UV damage sites, that both prevent local mitotic phosphorylations on histone H3 serine residues. Functionally, this chromatin-marking pathway drives the asymmetric segregation of UV damage in the cell progeny with consequences on daughter cell fate. We propose that this mechanism may help preserve the integrity of stem cell compartments during asymmetric cell divisions.

Trained immunity of synovial macrophages is associated with exacerbated joint inflammation and damage after Staphylococcus aureus infection.

Investigate whether and which synoviocytes would acquire trained immunity characteristics that could exacerbate joint inflammation following a secondary Staphylococcus aureus infection. Lipopolysaccharide (LPS) and S. aureus were separately or double injected (21 days of interval) into the tibiofemoral joint cavity of male C57BL/6 mice. At different time points after these stimulations, mechanical nociception was analyzed followed by the analysis of signs of inflammation and damage in the affected joints. The trained immunity markers, including the glycolytic and mTOR pathway, were analyzed in whole tissue or isolated synoviocytes. A group of mice was treated with Rapamycin, an mTOR inhibitor before LPS or S. aureus stimulation. The double LPS - S. aureus hit promoted intense joint inflammation and damage compared to single joint stimulation, including markers in synoviocyte activation, production of proinflammatory cytokines, persistent nociception, and bone damage, despite not reducing the bacterial clearance. The double LPS - S. aureus hit joints increased the synovial macrophage population expressing CX3CR1 alongside triggering established epigenetic modifications associated with trained immunity events in these cells, such as the upregulation of the mTOR signaling pathway (p-mTOR and HIF1α) and the trimethylation of histone H3. Mice treated with Rapamycin presented reduced CX3CR1+ macrophage activation, joint inflammation, and bone damage. There is a trained immunity phenotype in CX3CR1+ synovial macrophages that contributes to the exacerbation of joint inflammation and damage during septic arthritis caused by S. aureus.

Quantification of Histone H1 Subtypes Using Targeted Proteomics.

Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one piece of evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry-based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines, HeLa, K562, and T47D, showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. In CML, the first line of therapy is a tyrosine kinase inhibitor, imatinib. Our preliminary data revealed differences in the H1 complement in CML patients between imatinib responders and non-responders. These results support further research to determine if the H1 content or subtype composition could help predict imatinib response.

Liraglutide alleviates ferroptosis in renal ischemia reperfusion injury via inhibiting macrophage extracellular trap formation

Background and purposeRenal transplantation and other conditions with transiently reduced blood flow is major cause of renal ischemia/reperfusion injury (RIRI), a therapeutic challenge clinically. This study investigated the role of liraglutide in ferroptosis-associated RIRI via macrophage extracellular traps (METs). MethodsAnimal model with RIRI was established in C57BL/6J mice. A total of 72 C57BL/6J mice were used with 8 mice per group. Primary tubular epithelium was co-culture with RAW264.7 under hypoxia/reoxygenation (H/R) condition to mimic in vitro. Liraglutide was administrated into mice and cells. Extracellular DNA, neutrophil elastase and myeloperoxidase in serum and supernatant of cell medium were collected for measuring METs. F4/80 and citH3 were labeled to show METs. ResultsLiraglutide relieved RIRI and ferroptosis in vivo, and inhibited renal I/R-induced METs both in vivo and in vitro. F4/80 and citrullinated histone H3 (citH3) were highly co-localized after RIRI. Liraglutide attenuated the co-localization of citH3 and F4/80. Expressions of M2 markers were enhanced whereas these of M1 markers suppressed during liraglutide treatment in RIRI. Phosphorylation of signal transducer and activator of transcription (STAT)1, 3 and 6 were increased in RIRI mice and H/R-induced RAW264.7. However, liraglutide decreased phosphorylation of STAT1 and increased phosphorylation of STAT3 and STAT6. STAT3/6 inhibition reversed liraglutide-inhibited M1 polarization, extracellular traps and ferroptosis. ConclusionLiraglutide inhibited ferroptosis-induced renal dysfunction since it skewed macrophage polarization into M2 phenotype that interfered the formation of extracellular traps based on STAT3/6 pathway during RIRI. Liraglutide was proposed to be used for RIRI clinical treatment.

E74-like factor 4 promotes the proliferation, invasion, and migration of colorectal cancer cells via long non-coding RNA integrin subunit beta 8 antisense RNA 1-mediated histone H3 lysine 27 trimethylation modification.

Colorectal cancer (CRC) is a common malignancy in the gastrointestinal tract. The main objective of this study is to explore the potential mechanisms of E74-like factor 4 (ELF4) in CRC progression, providing a novel therapeutic target for CRC treatment. CRC cells and normal control cells were cultured. Levels of ELF4/long non-coding RNA integrin subunit beta 8 antisense RNA 1 (LncRNA ITGB8-AS1)/claudin-23 (CLDN23) were detected by real-time quantitative polymerase chain reaction or Western blot assay. ELF4 siRNA, ITGB8-AS1 pcDNA3.1, or CLDN23 siRNA were transfected into cells. Cell proliferation, migration, and invasion were evaluated. The enrichment of ELF4 on the ITGB8-AS1 promoter was detected. Dual-luciferase assay was employed to assess the binding between ELF4 and the ITGB8-AS1 promoter. RNA pull-down and RNA immunoprecipitation assays were conducted to investigate the binding between ITGB8-AS1 and enhancer of zeste homolog 2 (EZH2). The binding of EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to the CLDN23 promoter was detected. ELF4 and ITGB8-AS1 were upregulated in CRC cells, while CLDN23 was downregulated. Knockdown of ELF4 inhibited cell proliferation, invasion, and migration. Mechanistically, ELF4 transcriptionally activated ITGB8-AS1 and promoted the binding between ITGB8-AS1 and EZH2. EZH2 catalyzed H3K27me3 modification on the CLDN23 promoter, leading to decreased CLDN23 expression. Overexpression of ITGB8-AS1 or downregulation of CLDN23 could reduce the inhibitory effects of silencing ELF4 on CRC cell proliferation, migration, and invasion. ELF4 accelerates CRC progression through the ITGB8-AS1/CLDN23 axis, providing new therapeutic targets for CRC.

WDR5 Binding to Histone Serotonylation Is Driven by an Edge-Face Aromatic Interaction with Unexpected Electrostatic Effects.

Histone serotonylation has emerged as a key post-translational modification. WDR5 preferentially binds to serotonylated histone 3 (H3), and this binding event has been associated with tumorigenesis. Herein, we utilize genetic code expansion, structure-activity relationship studies, and computation to study an edge-face aromatic interaction between WDR5 Phe149 and serotonin on H3 that is key to this protein-protein interaction. We find experimentally that this edge-face aromatic interaction is unaffected by modulating the electrostatics of the face component but is weakened by electron-withdrawing substituents on the edge component. Overall, these results elucidate that this interaction is governed by van der Waals forces as well as electrostatics of the edge ring, a result that clarifies discrepancies among previous theoretical models and model system studies of this interaction type. This is the first evaluation of the driving force of an edge-face aromatic interaction at a protein-protein interface and provides a key benchmark for the nature of these understudied interactions that are abundant in the proteome.

The role of trimethylation on histone H3 lysine 27 (H3K27me3) in temozolomide resistance of glioma

Temozolomide (TMZ) is the first-line chemotherapeutic agent for malignant glioma, but its resistance limited the benefits of the treated patients. In this study, the role and significance of trimethylation of histone H3 lysine 27 (H3K27me3) in TMZ resistance were investigated. Data from twenty advanced glioma patients were collected, and their pathological samples were analyzed for H3K27me3 levels. TMZ sensitivity was compared between glioma cells U87 and TMZ-resistant cells U87TR, with H3K27me3 levels determined in both cells. The effects of H3K27me3 demethylases inhibitor GSK-J4, combined with TMZ, were assessed on the proliferation and migration of U87TR cells. The results indicated that a high level of H3K27me3 predicts longer disease free survival (DFS) and overall survival (OS) in glioma patients receiving TMZ treatment. The H3K27me3 level was lower in U87TR cells compared to U87 cells. GSK-J4 increased the H3K27me3 level in U87TR cells and decreased their resistance to TMZ. In summary, this study identified a novel marker of TMZ resistance in glioma and provided a new strategy to address this challenge. These findings are significant for improving the clinical treatment of glioma in the future.

Decreased NETosis-related regulators in neuromyelitis optica spectrum disorders after plasma exchange

ObjectivesTo investigate the impact of plasma exchange (PLEX) on NETosis-related regulators and their correlation with neurological improvement in NMOSD patients. MethodsTwelve aquaporin-4 antibodies seropositive NMOSD patients were enrolled. NETosis-related regulators (myeloperoxidase [MPO], citrullinated histone H3 [CIT-H3], peptidyl arginine deiminase 4 [PAD4], neutrophil elastase [NE], CD64), pro-inflammatory cytokines (IL-1, IL-6, IL-12, TNF-α) and anti-inflammatory cytokines (IL-10, TGF-β1) were quantitatively assessed before and after PLEX treatment. Clinical assessments included expanded disability status scale (EDSS) and visual outcome scale (VOS) scores. ResultsFollowing PLEX, all patients showed symptom improvement, with 66.7 % achieving marked-to-moderate improvement (MMI) at 3 months. Key regulators, such as MPO, CIT-H3, PAD4, NE, and pro-inflammatory cytokines such as IL-1, IL-6, IL-12, and TNF-α, exhibited a statistically significant decrease immediately after the initial PLEX session (P < 0.05). Furthermore, CD64 levels demonstrated a substantial decline after the second PLEX session (P < 0.05). Conversely, the levels of anti-inflammatory cytokines, including IL-10 and TGF-β1, displayed an ascending trend post-PLEX. In clinical relevance analysis, among patients who reached MMI, the reductions in MPO, IL-1, and IL-6 exhibited statistically significant differences when compared to patients in the mild-to-no improvement group (P < 0.05). Pearson correlation analysis revealed that the percentage reduction in IL-6 levels after PLEX was positively correlated with the percentage reduction in patient EDSS/VOS scores (r = 0.638, P < 0.05). ConclusionsThis study highlights that reduced levels of NETosis-related regulators after PLEX contribute to clinical improvement, suggesting the potential involvement of NETosis in the acute neurological impairment observed in NMOSD.

Identification of Cercospora spp. on corn in North America and baseline flutriafol fungicide sensitivity.

Gray leaf spot (GLS) is an important corn disease reportedly caused by Cercospora zeae-maydis and C. zeina. Recently, flutriafol, a demethylation inhibitor (azole) fungicide received EPA registration as Xyway® LFR®, a product that is applied at planting for management of fungal diseases in corn, including suppression of GLS. In this study, 448 Cercospora spp. isolates were collected in 2020 and 2021 from symptomatic corn leaf samples submitted from the United States and Ontario, Canada. The Cercospora spp. were identified using multi-locus genotyping of the internal transcribe spacer (ITS), elongation factor 1-α (EF1), calmodulin (CAL), histone H3 (HIS), and actin (ACT) gene. Based on the multi-locus phylogenetic analyses, six species were identified; C. cf. flagellaris (n = 77), C. kikuchii (n = 4), C. zeae-maydis (n = 361), Cercospora sp. M (n = 2), Cercospora sp. Q (n = 1), and Cercospora sp. T (n = 3). In subsequent pathogenicity tests using selected isolates from each of these species, only C. zeae-maydis resulted in symptoms on corn with no disease symptoms observed after inoculation with C. cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T. While disease symptoms were observed on soybean following inoculation with C. cf. flagellaris, C. kikuchii, and Cercospora sp. Q, but not the other three species. Fungicide sensitivity of Cercospora spp. to flutriafol was assessed using a subset of 340 isolates. The minimum inhibitory concentration (MIC) to inhibit the growth of Cercospora spp. completely was determined based on growth of each species on flutriafol-amended clarified V8 agar at nine concentrations. The EC50 was also calculated from the same trial by measuring relative growth as compared to the non-amended control. Cercospora zeae-maydis was sensitive to flutriafol with mean MIC values of 2.5 µg/mL and EC50 values ranging from 0.016 to 1.020 µg/mL with a mean of 0.346 µg/mL. Cercospora cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T had mean EC50 values of 1.25 µg/mL, 7.14 µg/mL, 2.48 µg/mL, 1.81 µg/mL, and 2.24 µg/mL respectively. These findings will assist in monitoring the sensitivity to the flutriafol fungicide in Cercospora spp. populations.

Effect of Epigenetic Inhibitors on Adipogenesis in Human Periodontal Ligament Stem Cells

Histone H3 lysine 9 acetylation is a crucial epigenetic mark in mesenchymal stem cell differentiation processes like adipogenesis. This study aimed to evaluate the effect of epigenetic inhibitors on adipogenesis in human periodontal ligament stem cells, using histone deacetylase inhibitors such as valproic acid and trichostatin A. The cells were treated independently with 8 mM valproic acid and 100 nM trichostatin A for 72 hours to inhibit class I histone deacetylases. Subsequently, the cells underwent adipogenic induction for 28 days. Morphology and intracellular lipid deposition were analyzed using Oil-Red oil staining. Protein extracts were prepared on days 0 and 28 to analyze histone H3 lysine 9 acetylation levels via Western blot. Our results demonstrated that cells treated with either VPA or TSA showed, on average, a 1.275-fold increase in lipid deposition during the adipogenic process, with no significant differences between the treatments compared to the control group. Further, by day 28, histone H3 lysine 9 acetylation levels were 1.43 times higher in cells treated with valproic acid and 2.52 times higher in those treated with trichostatin A.These findings suggest that the inhibitors could have differential effects on chromatin remodeling in different regions by increasing acetylation, contributing indistinctly to lipid deposition due to the greater expression of genes associated with the adipogenic phenotype.

The Natural HASPIN Inhibitor Coumestrol Suppresses Intestinal Polyp Development, Cachexia, and Hypogonadism in a Mouse Model of Familial Adenomatous Polyposis (ApcMin/+).

(1) Background: HASPIN kinase is involved in regulating spindle function and chromosome segregation, as well as phosphorylating histone H3 at Thr3 in mitotic cells. Several HASPIN inhibitors suppress cancer cell proliferation. It was recently reported that coumestrol from bean sprouts inhibits HASPIN, and a cultivation method for bean sprouts containing large amounts of coumestrol has been established. Here, we showed the effects of bean sprout ingestion on intestinal polyp development, cachexia, and hypogonadism in a mouse model of familial adenomatous polyposis (ApcMin/+). (2) Methods: ApcMin/+ mice were randomized into control and treatment groups. Mice in the control group were given the standard diet, while those in the treatment group were given the same standard diet with the addition of 15% bean sprouts. Treatments were commenced at 7 weeks old and analyses were performed at 12 weeks old. (3) Results: ingesting bean sprouts suppressed the development of intestinal polyps, cachexia, and hypogonadism, and also increased serum levels of testosterone in male wild-type and ApcMin/+ mice. (4) Conclusions: ingesting bean sprouts helps prevent cancer and increases serum levels of testosterone in a mouse model. These results are expected to be applicable to humans.

EZH2 Inhibition by DS3201 Triggers the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle and Potentiates the Effects Induced by SAHA in Primary Effusion Lymphoma Cells.

Primary Effusion Lymphoma (PEL) cells carry Kaposi's sarcoma-associated herpesvirus (KSHV) in a latent state, except for a small number of cells in which the virus replicates to ensure its persistence into the infected host. However, the lytic cycle can be reactivated in vitro by exposing these lymphoma cells to various treatments, leading to cell lysis. To restrict viral antigen expression, KSHV induces repressive epigenetic changes, including DNA methylation and histone modifications. Among the latter, histone deacetylation and tri-methylation of Histone H3 lisyne-27 (H3K27me3) have been reported to play a role. Here, we found that the inhibition of H3K27 tri-methylation by valemetostat DS3201 (DS), a small molecule that inhibits Enhancer of Zeste Homolog 2 (EZH2) methyltransferase, induced the KSHV lytic cycle in PEL cells, and that this effect involved the activation of the wtp53-p21 axis and autophagic dysregulation. DS also potentiated the lytic cycle activation mediated by the Histone deacetylases (HDAC) inhibitor Suberoylanilide hydroxamic acid (SAHA) and reinforced its cytotoxic effect, suggesting that such a combination could be used to unbalance the latent/lytic cycle and further impair the survival of PEL cells.

Pterocarya hupehensis Skan total flavones ameliorate rheumatoid arthritis in rats by suppressing formation of neutrophil extracellular traps

To investigate the therapeutic mechanism of Pterocarya hupehensis Skan total flavonoids (PHSTF) for rheumatoid arthritis (RA). Twenty-five male SD rats were randomly divided into normal control group, RA model group, PHSTF treatment (45 and 90 mg/kg) groups, and Tripterygium glycosides (TPG) tablet (10 mg/kg) group (n=5). Except for those in the normal control group, all the rats were subjected to collagen-induced arthritis (CIA) modeling using a secondary immunization method, after which PHSTF and TPG were administered via gavage once daily for 4 weeks. After the treatments, serum levels of TNF-α and IL-1β were measured using ELISA, and ankle joint pathologies were assessed with HE staining; the expression of citrullinated histone H3 (Cit-H3), a neutrophil extracellular trap (NET) marker, in the ankle joints was evaluated with immunohistochemistry. In primary cultures of rat peripheral blood neutrophils stimulated with phorbol ester (PMA), the effects of PHSTF (100 and 200 μg/mL) on the expressions of Cit-H3, peptidylarginine deiminase 4 (PADI4), neutrophil elastase (NE), and myeloperoxidase (MPO) were examined with Western blotting; immunofluorescence assay was used to observe Cit-H3 expression and NET formation in the cells. In the CIA rat models, PHSTF significantly alleviated ankle swelling, decreased serum levels of TNF-α and IL-1β, improved histopathological changes in the ankle joints, and reduced Cit-H3 expression in both the serum and ankle joint cartilage. In the isolated rat neutrophils, PHSTF showed no significant effect on cell viability but strongly inhibited PMA-induced NET release. PHSTF can alleviate RA by inhibiting the formation of NETs.

Multiplexed spatial mapping of chromatin features, transcriptome, and proteins in tissues.

The phenotypic and functional states of a cell are modulated by a complex interactive molecular hierarchy of multiple omics layers, involving the genome, epigenome, transcriptome, proteome, and metabolome. Spatial omics approaches have enabled the capture of information from different molecular layers directly in the tissue context. However, current technologies are limited to map one to two modalities at the same time, providing an incomplete representation of cellular identity. Such data is inadequate to fully understand complex biological systems and their underlying regulatory mechanisms. Here we present spatial-Mux-seq, a multi-modal spatial technology that allows simultaneous profiling of five different modalities, including genome-wide profiles of two histone modifications and open chromatin, whole transcriptome, and a panel of proteins at tissue scale and cellular level in a spatially resolved manner. We applied this technology to generate multi-modal tissue maps in mouse embryos and mouse brains, which discriminated more cell types and states than unimodal data. We investigated the spatiotemporal relationship between histone modifications, chromatin accessibility, gene and protein expression in neuron differentiation revealing the relationship between tissue organization, function, and gene regulatory networks. We were able to identify a radial glia spatial niche and revealed spatially changing gradient of epigenetic signals in this region. Moreover, we revealed previously unappreciated involvement of repressive histone marks in the mouse hippocampus. Collectively, the spatial multi-omics approach heralds a new era for characterizing tissue and cellular heterogeneity that single modality studies alone could not reveal.

Characterization of Neutrophil Functional Responses to SARS-CoV-2 Infection in a Translational Feline Model for COVID-19.

There is a complex interplay between viral infection and host innate immune response regarding disease severity and outcomes. Neutrophil hyperactivation, including excessive release of neutrophil extracellular traps (NETs), is linked to exacerbated disease in acute COVID-19, notably in hospitalized patients. Delineating protective versus detrimental neutrophil responses is essential to developing targeted COVID-19 therapies and relies on high-quality translational animal models. In this study, we utilize a previously established feline model for COVID-19 to investigate neutrophil dysfunction in which experimentally infected cats develop clinical disease that mimics acute COVID-19. Specific pathogen-free cats were inoculated with SARS-CoV-2 (B.1.617.2; Delta variant) (n = 24) or vehicle (n = 6). Plasma, bronchoalveolar lavage fluid, and lung tissues were collected at various time points over 12 days post-inoculation. Systematic and temporal evaluation of the kinetics of neutrophil activation was conducted by measuring markers of activation including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (citH3) in SARS-CoV-2-infected cats at 4 and 12 days post-inoculation (dpi) and compared to vehicle-inoculated controls. Cytokine profiling supported elevated innate inflammatory responses with specific upregulation of neutrophil activation and NET formation-related markers, namely IL-8, IL-18, CXCL1, and SDF-1, in infected cats. An increase in MPO-DNA complexes and cell-free dsDNA in infected cats compared to vehicle-inoculated was noted and supported by histopathologic severity in respiratory tissues. Immunofluorescence analyses further supported correlation of NET markers with tissue damage, especially 4 dpi. Differential gene expression analyses indicated an upregulation of genes associated with innate immune and neutrophil activation pathways. Transcripts involved in activation and NETosis pathways were upregulated by 4 dpi and downregulated by 12 dpi, suggesting peak activation of neutrophils and NET-associated markers in the early acute stages of infection. Correlation analyses conducted between NET-specific markers and clinical scores as well as histopathologic scores support association between neutrophil activation and disease severity during SARS-CoV-2 infection in this model. Overall, this study emphasizes the effect of neutrophil activation and NET release in SARS-CoV-2 infection in a feline model, prompting further investigation into therapeutic strategies aimed at mitigating excessive innate inflammatory responses in COVID-19.

The Drosophila maternal-effect gene abnormal oocyte (ao) does not repress histone gene expression.

The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created two new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that ao exhibits maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. We also confirm that the Ao protein localizes to the histone locus bodies in ovaries. Our data also suggest that ao genetically interacts with the histone genes and heterochromatin, as previously suggested. However, contrary to prior findings, we find that ao does not repress core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown.

Cyclic stretch enhances neutrophil extracellular trap formation

BackgroundNeutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries.ResultsUsing this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization.ConclusionsIn summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target.

A novel dual-epigenetic inhibitor enhances recombinant monoclonal antibody expression in CHO cells

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 μM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells.Key points• HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells.• By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased.• It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

Nuclear factor-Y-polycomb repressive complex2 dynamically orchestrates starch and seed storage protein biosynthesis in wheat.

The endosperm in cereal grains is instrumental in determining grain yield and seed quality, as it controls starch and seed storage protein (SSP) production. In this study, we identified a specific nuclear factor-Y (NF-Y) trimeric complex in wheat (Triticum aestivum L.), consisting of TaNF-YA3-D, TaNF-YB7-B, and TaNF-YC6-B, and exhibiting robust expression within the endosperm during grain filling. Knockdown of either TaNF-YA3 or TaNF-YC6 led to reduced starch but increased gluten protein levels. TaNF-Y indirectly boosted starch biosynthesis genes by repressing TaNAC019, a repressor of cytosolic small ADP-glucose pyrophosphorylase 1a (TacAGPS1a), sucrose synthase 2 (TaSuS2), and other genes involved in starch biosynthesis. Conversely, TaNF-Y directly inhibited the expression of Gliadin-γ-700 (TaGli-γ-700) and low molecular weight-400 (TaLMW-400). Furthermore, TaNF-Y components interacted with SWINGER (TaSWN), the histone methyltransferase subunit of Polycomb repressive complex 2 (PRC2), to repress TaNAC019, TaGli-γ-700, and TaLMW-400 expression through trimethylation of histone H3 at lysine 27 (H3K27me3) modifications. Notably, weak mutation of FERTILIZATION INDEPENDENT ENDOSPERM (TaFIE), a core PRC2 subunit, reduced starch but elevated gliadin and LMW-GS contents. Intriguingly, sequence variation within the TaNF-YB7-B coding region was linked to differences in starch and SSP content. Distinct TaNF-YB7-B haplotypes affect its interaction with TaSWN-B, influencing the repression of targets like TaNAC019 and TaGli-γ-700. Our findings illuminate the intricate molecular mechanisms governing TaNF-Y-PRC2-mediated epigenetic regulation for wheat endosperm development. Manipulating the TaNF-Y complex holds potential for optimizing grain yield and enhancing grain quality.