Histone H3 Acetylation Levels Research Articles

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.

Epigenetic alternations in the SYP and DLG4 genes due to low-level methylmercury exposure during neuronal differentiation in vitro.

Methylmercury (MeHg) is an environmental toxin known to damage the central nervous system. When pregnant women ingest seafood, which may contain accumulated MeHg, fetal development may be affected. The embryonic period, a time of major epigenetic change, is susceptible to epigenetic disruptions due to chemical exposure. Therefore, understanding the molecular mechanism underlying MeHg's effects on neuronal development requires consideration of epigenetic factors. In this study, we investigated epigenetic modifications in the synaptophysin (SYP) and discs large MAGUK scaffold protein 4 (DLG4) genes. LUHMES cells were exposed to 1 nM MeHg for 6 days during days 2-8 of neural differentiation. MeHg exposure significantly reduced the number of spikes observed on day 16 of differentiation. Both mRNA and protein expression levels of SYP and DLG4 were significantly decreased by MeHg exposure. Additionally, MeHg treatment reduced acetyl histone H3 levels associated with transcriptional activity in the SYP gene while increasing histone H3 lysine 27 tri-methylation (H3K27me3) levels related to transcriptional repression. Conversely, regarding the DLG4 gene, MeHg exposure increased H3K27me3 levels. Differential changes in DNA methylation (high and low methylation states) were observed in the SYP and DLG4 genes due to MeHg exposure depending on CpG site position. In conclusion, this study suggests that epigenetic changes, particularly histone modifications, contribute to decreased MeHg exposure-induced SYP and DLG4 expression during neuronal differentiation.

Extracellular C1qbp inhibits myogenesis by suppressing NFATc1

Aging and lack of exercise are the most important etiological factors for muscle loss. We hypothesized that new factors that contribute to muscle loss could be identified from ones commonly altered in expression in aged and exercise-limited skeletal muscles. Mouse gastrocnemius muscles were subjected to mass spectrometry-based proteomic analysis. The muscle proteomes of hindlimb-unloaded and aged mice were compared to those of exercised and young mice, respectively. C1qbp expression was significantly upregulated in the muscles of both hindlimb-unloaded and aged mice. In vitro myogenic differentiation was not affected by altering intracellular C1qbp expression but was significantly suppressed upon recombinant C1qbp treatment. Additionally, recombinant C1qbp repressed the protein level but not the mRNA level of NFATc1. NFATc1 recruited the transcriptional coactivator p300, leading to the upregulation of acetylated histone H3 levels. Furthermore, NFATc1 silencing inhibited p300 recruitment, downregulated acetylated histone H3 levels, and consequently suppressed myogenic differentiation. The expression of C1qbp was inversely correlated with that of NFATc1 in the gastrocnemius muscles of exercised or hindlimb-unloaded, and young or aged mice. These findings demonstrate a novel role of extracellular C1qbp in suppressing myogenesis by inhibiting the NFATc1/p300 complex. Thus, C1qbp can serve as a novel therapeutic target for muscle loss.

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.

Anatomical distribution of histone H3 acetylation in human hippocampus

In recent years, the role of histone acetylation in the brain has been studied from different aspects. However, the anatomical distribution of histone H3 acetylation in the human hippocampus and its potential relevance to triple synaptic circuits are unknown. As one of epigenetic remodeling ways, the modification of histones is involved in multiple aspects of neuronal function and development and is a key process in the onset and development of Alzheimer’s disease (AD). In our study, we compared acetylation levels of histone H3 at different regions of the hippocampus in AD and non-AD patients. We found that histone H3 acetylation can be detected in the dentate gyrus (DG), CA4, CA3, CA2, CA1, and lower Toya regions of the human hippocampus. The highest degree of acetylation in the hippocampus is in DG, and the level of acetylation changes gradually and systematically along the triple synaptic circuit. Besides, there were no significant differences in histone acetylation between AD and non-AD groups.

A Novel Class I HDAC Inhibitor, AW01178, Inhibits Epithelial-Mesenchymal Transition and Metastasis of Breast Cancer.

Epithelial-mesenchymal transition (EMT) refers to the transformation of polar epithelial cells into motile mesenchymal cells under specific physiological or pathological conditions, thus promoting the metastasis of cancer cells. Epithelial cadherin (E-cadherin) is a protein that plays an important role in the acquisition of tumor cell motility and serves as a key EMT epithelial marker. In the present study, AW01178, a small-molecule compound with potential therapeutic efficacy, was identified via in-cell Western high-throughput screening technology using E-cadherin as the target. The compound induced the upregulation of E-cadherin at both mRNA and protein levels and inhibited the EMT of breast cancer cells in vitro as well as metastasis in vivo. Mechanistically, AW01178 is a novel benzacetamide histone deacetylase inhibitor (HDACi) mainly targeting class I histone deacetylases. AW01178 promoted the transcription and expression of E-cadherin through enhancing the acetylation level of histone H3 in the E-cadherin promoter region, thereby inhibiting the metastasis of breast cancer cells. The collective findings support the potential utility of the novel HDACi compound identified in this study, AW01178, as a therapeutic drug for breast cancer and highlight its value for the future development of HDACi structures as anticancer drugs.

Epigenetic impact of hypothyroidism on the functional differentiation of the mammary gland in rats

Mammary gland (MG) lactogenic differentiation involves epigenetic mechanisms. We have previously shown that hypothyroidism (HypoT) alters the MG transcriptome in lactation. However, the role of thyroid hormones (T3 and T4 a. k.a. THs) in epigenetic differentiation of MG is still unknown. We used a model of post-lactating HypoT rats to study in MG: a) Methylation and expression level of Gata3, Elf5, Stat6, Stat5a, Stat5b; b) Expression of Lalba, IL-4Rα and Ncoa1 mRNA; c) Histone H3 acetylation and d) Estrogen and progesterone concentration in serum. HypoT increases the estrogen serum level, decreases the progesterone level, promotes methylation of Stat5a, Stat5b and Stat6, decreasing their mRNA level and of its target genes (Lalba and IL-4Rα) and increases the Ncoa1 mRNA expression and histone H3 acetylation level. Our results proved that HypoT alters the post-lactation MG epigenome and could compromise mammary functional differentiation.

Sea cucumber intestinal osteotropic peptide promotes tibial fracture healing in osteoporotic mice by promoting glutamine metabolism

Sea cucumber (Stichopus japonicus) intestinal high-value-added compounds have not been fully used in sea cucumber processing. Here, the osteotropic effects of sea cucumber intestinal peptides (SCIP) on tibial fractures in osteoporotic mice were investigated. Mice were bilaterally ovariectomized to model osteoporosis and achieved open fracture surgery on the right tibia. The results exhibited that SCIP intervention significantly elevated localized callus and total glutamine levels after fracture in osteoporotic mice. SCIP intervention enhanced the level of acetyl coenzyme A-mediated histone H3 acetylation by up-regulation of the GLS1-GLUD1 axis, thereby significantly promoting the expression of genes specific to chondrocytes. Also, it enhanced aspartate-mediated purine and pyrimidine synthesis by up-regulation of the GLS1-GOT2 axis, thereby significantly promoting the proliferation of chondrocytes. Moreover, it enhanced glutathione-mediated antioxidant damage by upregulating the GLS1-GCLC axis, thereby significantly rescuing the pathological apoptosis of hypertrophic chondrocytes. In conclusion, SCIP promoted chondrocyte generation, proliferation, mineralization, and bone remodeling through glutamine metabolism, thus facilitating fracture healing in osteoporotic mice.

Tinostamustine (EDO-S101), an Alkylating Deacetylase Inhibitor, Enhances the Efficacy of Daratumumab in Multiple Myeloma by Upregulation of CD38 and NKG2D Ligands.

Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.

Comparative neuroprotective effects of royal jelly and its unique compound 10-hydroxy-2-decenoic acid on ischemia-induced inflammatory, apoptotic, epigenetic and genotoxic changes in a rat model of ischemic stroke

ABSTRACT Objectives This study aimed to compare the efficacy of royal jelly (RJ) and its major fatty acid 10-hydroxy-2-decenoic acid (10-HDA) on ischemic stroke-related pathologies using histological and molecular approaches. Methods Male rats were subjected to middle cerebral artery occlusion (MCAo) to induce ischemic stroke and were supplemented daily with either vehicle (control group), RJ or 10-HDA for 7 days starting on the day of surgery. On the eighth day, rats were sacrificed and brain tissue and blood samples were obtained to analyze brain infarct volume, DNA damage as well as apoptotic, inflammatory and epigenetic parameters. Results Both RJ and 10-HDA supplementation significantly reduced brain infarction and decreased weight loss when compared to control animals. These effects were associated with reduced levels of active caspase-3 and PARP-1 and increased levels of acetyl-histone H3 and H4. Although both RJ and 10-HDA treatments significantly increased acetyl-histone H3 levels, the effect of RJ was more potent than that of 10-HDA. RJ and 10-HDA supplementation also alleviated DNA damage by significantly reducing tail length, tail intensity and tail moment in brain tissue and peripheral lymphocytes, except for the RJ treatment which tended to reduce tail moment in lymphocytes without statistical significance. Conclusions Our findings suggest that neuroprotective effects of RJ in experimental stroke can mostly be attributed to 10-HDA.

Melatonin modulates lipopolysaccharides-induced inflammatory response and maintains circadian rhythm associated with histone H3 acetylation in bovine mammary epithelial cells

Lipopolysaccharides (LPS) can disrupt the barrier function of mammary epithelial cells, leading to mastitis. The protective effects of melatonin (MT) have been demonstrated against various infections, but the underlying mechanism remains unclear in LPS-induced bovine mastitis. This study, therefore, aimed to investigate the modulatory effect of melatonin (MT) on LPS-induced inflammation in bovine mammary epithelial cells (BMECs), its influence on the disruption of circadian rhythms triggered by this inflammation, and the potential underlying molecular mechanisms. BMECs extracted from lactating cows underwent one of 4 treatments in triplicates for 12 h simultaneously: Control (CON), LPS (exposed to LPS at 1 μg/mL), MT (exposed to MT at 40 μmol/L), or LPS + MT (LMT, exposed to both LPS at 1 μg/mL and MT at 40 μmol/L). The flow cytometry results demonstrated that MT reduced the ratios of cells in the G0/G1 phase but increased those in the G2/M phase (P < 0.05). Additionally, MT inhibited p65 phosphorylation levels, leading to a decreased cellular inflammatory response (P < 0.05). Treatment with MT elevated the acetylation levels of histone H3 in BMECs compared to the LPS treatment (P < 0.05). The circadian rhythm analysis revealed a significant increase in the gene expression levels of BMAL1, PER1, CRY1, and CRY2 at the 12 h mark in LMT compared to the LPS treatment (P < 0.05). Additionally, this study assessed the fluctuations in clock-related genes PER2 and CLOCK over 24 h under various treatment conditions. The results demonstrated that BMECs exposed to LMT treatment exhibited a significant increase in the expression levels of CLOCK and PER2 genes from 4 to 24 h in comparison to the LPS treatment (P < 0.05). Moreover, cells in the LMT treatment displayed a more regular circadian rhythm cycle compared to those in the LPS treatment. Overall, the data suggest that MT could alleviate the inflammatory response in BMECs by upregulating histone H3 acetylation, thereby maintaining the physiological circadian rhythm of BMECs.

Systemic histone deacetylase inhibition ameliorates the aberrant responses to acute stress in socially isolated male mice.

Adverse experiences in early life can induce maladaptive responses to acute stress in later life. Chronic social isolation during adolescence is an early life adversity that can precipitate stress-related psychiatric disorders. We found that male mice after 8weeks of adolescent social isolation (SI) have markedly increased aggression after being exposed to 2h of restraint stress (RS), which was accompanied by a significant increase of AMPA receptor- and NMDA receptor-mediated synaptic transmission in prefrontal cortex (PFC) pyramidal neurons of SIRS males. Compared to group-housed counterparts, SIRS males exhibited a significantly decreased level of histone H3 acetylation in PFC. Systemic administration of class I histone deacetylase inhibitors, romidepsin or MS-275, ameliorated the aggressive behaviour, as well as general social interaction deficits, of SIRS males. Electrophysiological recordings also found normalization of PFC glutamatergic currents by romidepsin treatment of SIRS male mice. These results revealed an epigenetic mechanism and intervention avenue for aggression induced by chronic social isolation. KEY POINTS: Adolescent chronic social isolation can precipitate stress-related psychiatric disorders. A significant increase of glutamatergic transmission is found in the prefrontal cortex (PFC) of socially isolated male mice exposed to an acute stress (SIRS). Treatment with class I histone deacetylase (HDAC) inhibitors ameliorates the aggressive behaviour and social interaction deficits of SIRS males, and normalizes glutamatergic currents in PFC neurons. It provides an epigenetic mechanism and intervention avenue for aberrant stress responses induced by chronic social isolation.

Study of the role of evolutionary new enhancers in the development of the &lt;i&gt;corpus callosum&lt;/i&gt;

One important aspect of the mammalian brain is the exchange of information between neurons located in different hemispheres. This process evolved during the development of mammals. In marsupial (Marsupialia) and monotreme (Monotremata) mammals, the communication between hemispheres is facilitated through an enlarged anterior commissure. In placental (Eutheria) mammals, a new brain structure, the corpus callosum, emerged during the evolutionary process. The corpus callosum, comprising 80% of the brain’s commissural axons, is the largest commissure in the human body. The corpus callosum is a major contributor to the efficiency of higher neural activities, including memory, decision making, social interaction, and language. A possible explanation for the emergence of a novel structure for interhemispheric interaction is a change in the growth direction of neocortical axons during development. Changes in gene expression levels can regulate this process, which involves controlling axon growth and cell migration in the neocortex. A full comprehension of this process will enable the creation of new animal models for studying cortical malformations and the navigation of axons in the cerebral cortex leading to the formation of interhemispheric connections. Several enhancers were identified for protein-coding genes with differing expression patterns in neocortical cells of placental and non-placental (marsupial) mammals. Throughout the genomes of the house opossum (Monodelphis domestica) and the house mouse (Mus musculus), the acetylation levels of histone H3 on lysine 27 (H3K27ac) were compared. H3K27ac is considered an epigenetic marker for active gene enhancers. Then, a screening of candidate genes was performed to evaluate their localization and expression levels in the cortex during embryonic development. Thus, the Tbr1 gene was identified. Incorrect expression of this gene may result in changes to cortical development. The CRISPR/Cas9 system was combined with in utero electroporation to completely delete the active Tbr1 gene enhancer in developing neocortical cells of mouse embryos at day 14 of embryonic development. The impact of this enhancer deletion was then analyzed on the expression of Tbr1 in the upper layers of the cortex, as well as the direction of axon growth and neuronal migration on the 18th day of embryonic development. A significant reduction in Tbr1 expression was observed in the upper layers of the cortex after deletion of the active enhancer. Only 30% of the electroporated neurons retained Tbr1 expression. Moreover, a considerable delay in neuronal migration was observed in the subventricular zone (41% versus 17% in the control group) and in the upper layers of the cortex (20% versus 35% in the control group). However, the direction of axonal growth remained unchanged: callosal axons effectively crossed the midline and created the corpus callosum. Thus, expression of the evolutionary novel Tbr1 enhancer is important for neuronal migration during corticogenesis. However, its contribution to the development of the corpus callosum is not fully understood. A detailed analysis of the corpus callosum morphology post-enhancer deletion will be the subsequent step.

Histone acetylation is associated with pupal diapause in cotton bollworm, Helicoverpa armigera.

Diapause is an environmentally preprogrammed period of arrested development that is important to insect survival and population growth. Histone acetylation, an epigenetic modification, has several biological functions, but its role in agricultural pest diapause is unknown. In this study, we investigated the role of histone H3 acetylation in the diapause of Helicoverpa armigera. The histone H3 gene of H. armigera was cloned, and multiple sequence alignment of amino acids revealed that the potential lysine acetylation sites were highly conserved across species. Investigation of histone H3 acetylation levels in diapause- and nondiapause-type pupae showed that acetylation levels were down-regulated in diapause-type pupae and were lower in diapausing pupae compared to nondiapause pupae. By screening the genome, six histone acetyltransferase (HAT) and eight histone deacetylase (HDAC) genes responsible for antagonizing catalytic histone acetylation modifications were identified in H. armigera, and most of them exhibited different expression patterns between diapause- and nondiapause-type pupae. To elucidate the effect of histone H3 acetylation on diapause in H. armigera, the diapause pupae were injected with the histone acetylation activator trichostatin A (TSA). The results indicated that TSA injection increased the levels of histone H3 acetylation, causing the diapausing pupae to revert to development. Furthermore, transcriptome analysis revealed that 259 genes were affected by TSA injection, including genes associated with metabolism, resistance, and immunological responses. These results suggest that histone acetylation is inseparably related to the pupal diapause of H. armigera, which promises to be a potential target for pest control. © 2023 Society of Chemical Industry.

Discovery of benzamide-based PI3K/HDAC dual inhibitors with marked pro-apoptosis activity in lymphoma cells

Inhibition of PI3K and histone deacetylase (HDAC) activity simultaneously using a single molecule appears to be a promising approach for cancer treatment. Current PI3K/HDAC dual inhibitors commonly use hydroxamate moiety as zinc binding group, which lack HDAC isoform selectivity and have potential genotoxicity. In this study, a novel series of benzamide-based PI3K/HDAC dual inhibitors were rationally designed and synthesized. Representative compound PH14 showed potent inhibitory activity toward PI3Kα and HDAC3, with IC50 values of 20.3 nM and 24.5 nM, respectively. This was further supported by the blockage of AKT phosphorylation and an increase in acetylated histone H3 levels in Western blot study. The advantage of simultaneously targeting PI3Kα and HDAC is not only reflected in the significant antiproliferative activity, but also in its ability to promote the apoptosis in Jeko-1 cells. Moreover, PH14 had weak inhibitory effects on CYP450 enzymes and hERG. In the pharmacokinetic study, the administration of 1 mg/kg of PH14 the administration of 1 mg/kg of PH14 resulted in a t1/2 of 10 h and an AUC (0-∞) of 2772 h ng/mL. Our study may provide ideas for the further development of novel HDAC/PI3K dual inhibitors.

Epigenetic Reprogramming By Histone Deacetylase Inhibitors Improves CAR-T Cell Functions

Background: The exhaustion and diminished persistence of CAR-T cells primarily affect clinical efficacy and long-term outcomes. Epigenetic is an innate mechanism in regulating the biological function CAR-T cells. We previously dissected comprehensive dynamic changes of chromatin accessibility and transcription factors during CAR-T cell exhaustion (Jiang P, et al. Leukemia. 2022 Nov;36(11):2656-2668). Therefore, combined application with epigenetic drugs have much therapeutic potential. Methods: We initially performed an extensive screening among 370 epigenetic drugs to select candidates that could resist exhaustion and maintain phenotype of memory subsets, and thoroughly verify their effects on CAR-T cells in vitro. Next, we conducted multi-omics analysis comprising RNA-seq, ATAC-seq, and CUT&amp;Tag-seq to explore underlying regulative mechanisms of epigenetic manipulation by epi-drugs. In the end, we used preclinical B-ALL NSG mouse model to verify anti-tumor activities of drug-treated CAR-T cells in vivo. Results: Among those screened epigenetic drugs, we identified two histone deacetylase inhibitors (HDACi), Chidamide and M344, could best promote maintenance of central memory subsets and resistance to exhaustion of CAR-T cells, with enhanced anti-leukemia activities in vitro (Figure 1a-c). Chidamide and M344 inhibited enzymatic activity and expression of HDAC1, which elevated the acetylation level of histone H3, particularly at H3K27 in CD19 CAR-T cells. Genetic knock-down of HDAC1 could partially reflect the phenotypic mimicry by Chidamide and M344 on CAR-T cells. By combining multi-omics data from bulk RNA-seq, ATAC-seq, and H3K27ac-targeted CUT&amp;Tag-seq in Chidamide-or M344-treated CAR-T cells, we identified distinct patterns of epigenetic reprogramming, and enrichment of genes associated with exhaustion, as well as regulation of cellular differentiation and formation of memory subsets. In addition, analysis of motif and interaction network among transcription factors revealed altered regulation of gene transcription (data not shown). Finally, either Chidamide-pretreated CAR-T cells or in vivo administration of Chidamide after CAR-T cells infusion had exhibited enhanced and persistent tumor clearance in preclinical B-ALL NSG mouse model (Figure 1d). Conclusion: we conducted comprehensive screening of 370 epigenetic drugs and identified that administration of HDACi, particularly Chidamide and M344, could induce significantly enhanced anti-tumor function with maintenance of memory phenotype and reduced exhaustion in CAR-T cells through epigenetic reprogramming and altered regulation of gene transcription, which provided with basis and therapeutic targets for the rational combination of CAR-T cell therapy and epigenetic drugs in patients with hematological malignancies. Figure legends: Figure1. Screening of 370 epigenetic drugs identified M344 and Chidamide that enhanced maintenance of memory phenotype, resistance to exhaustion, and ability of tumor clearance in CAR-T cells. (a) The scatterplot of screening 370 compounds based on relative CD45RO +CD62L + proportion of central memory in treated CAR-T cell, showing of compared with DMSO control group by flow cytometry. Red indicates an increase in the proportion of memory CAR-T cells, while blue represents a decrease. (b) Frequency of co-expression of exhaustion-related inhibitory receptors (LAG-3, TIM-3, and PD-1) in DMSO or HDACi-treated groups. Data presented as mean ± SEM of independent donors (n=3 or more). (c) Flow cytometric analysis of central memory and effector memory subsets in CAR-T cells in each group. Data presented as mean ± SEM of independent donors (n=3 or more). (d) Bioluminescent imaging of tumor clearance in B-ALL NSG mouse. ‘X’ represents dead subject (end point). Survival of B-ALL NSG mice i.v. infused with T cells or treated CAR-T cells. Statistical analysis by Mantel-Cox test. Data presented as pooled from independent experiments (n=2).

ZMYM3 Mutations Cooperate with NOTCH1 Alterations, Reduce Histone H4 Acetylation and Promote Apoptosis Evasion in Chronic Lymphocytic Leukemia

Introduction: Recent next-generation sequencing (NGS) studies have identified up to 200 recurrently mutated genes in chronic lymphocytic leukemia (CLL), the vast majority of which appear mutated in &amp;lt;5% of CLL cases. Among the long list of CLL candidate driver genes is ZMYM3, a gene mutated in 2-4% of CLL patients. Since the impact of ZMYM3 mutations is unknown, we aimed to identify their clinical and biological consequences in CLL. Methods: 487 CLL patients were analyzed by targeted NGS using a customized panel of 54 CLL-related genes to characterize the mutational profile of ZMYM3 mutated patients ( ZMYM3MUT) and the clinical impact of these variants. In addition, we introduced ZMYM3 truncating mutations into the CLL-derived HG3 cell line with two genetic backgrounds: wild-type (WT), and NOTCH1 mutated cells ( NOTCH1MUT), to generate cells with either single ZMYM3MUT, NOTCH1MUT or combined ZMYM3MUTNOTCH1MUT. In these CLL models, we evaluated the impact of these alterations in gene expression, apoptosis, growth and DNA damage response (DDR). Results: A total of 32 ZMYM3 variants were identified in 30 CLL patients, most of which were loss-of-function mutations (24/32; 75%). These variants were distributed throughout the protein sequence, with a particular recurrent frameshift mutation occurring in 6 patients (p.P48fs) that was subsequently reproduced in our CLL model. Attending to their mutational profile, we identified that 70% (21/30) of ZMYM3MUT patients harbored mutations in the NOTCH pathway, either in NOTCH1 (60%) or in negative regulators such as MED12, FBXW7 and SPEN, suggesting a cooperation between ZMYM3 dysfunction and NOTCH1 signaling mutations in CLL evolution. Interestingly, ZMYM3MUT patients exhibited significantly shorter time to first treatment (TFT) than ZMYM3WT cases (median: 35 vs 52 months; p=0.010). Furthermore, ZMYM3 mutations shortened TFT of early stage CLL patients, including Binet A (48 vs 108 months, p=0.002) or Rai 0/1 (48 vs 91 months, p=0.016), as well as in low-risk cytogenetics del(13q) patients (median: 45 vs 93 months; p=0.033). We next addressed the biological implications of ZMYM3 mutations and their co-occurrence with NOTCH1 mutations in HG3 cells. First, we performed RNA-sequencing and analyzed the number of differentially expressed genes (DEGs) (|fold change|&amp;gt;2, adjusted p-value&amp;lt;0.05). Comparing with WT cells, we detected 155 DEGs in ZMYM3MUT and 83 DEGs in NOTCH1MUT, whereas ZMYM3MUTNOTCH1MUT cells showed 690 DEGs,indicating that the combination of both mutations profoundly dysregulate gene expression. Immune regulation, apoptosis and DNA damage response were some of the biological processes dysregulated by ZMYM3 mutations (p&amp;lt;0.05). Furthermore, considering the suggested role of ZMYM3 in chromatin remodeling (Puente et al, Nature. 2015) and that ZMYM3MUT cells showed altered expression of histone acetylation related genes (p&amp;lt;0.05), we identified that ZMYM3 mutations reduced global acetylation levels of histone H4 (p&amp;lt;0.05), which may underlie the ZMYM3-related changes in gene expression. To further define the functional effects of ZMYM3 mutations, we assessed in vitro their impact in the main dysregulated pathways identified by RNA-seq. First, we identified that ZMYM3 mutations impair DNA damage response, as reflected by the difficulties to arrest cell cycle in G2/M phase after irradiation. Moreover, we detected more γH2AX foci (p&amp;lt;0.05) in ZMYM3MUTcells, indicating persistent DNA damage; and fewer RAD51 and BRCA1 foci than WT cells (p&amp;lt;0.001) after irradiation, suggesting defective DNA damage response. In parallel, we identified that ZMYM3 mutations promote apoptosis evasion, especially in the context of NOTCH1 mutations (p&amp;lt;0.001), which led to enhanced growth of ZMYM3MUTNOTCH1MUT cells (p&amp;lt;0.05). Of note, ZMYM3MUT cells showed down-expression of caspases (-8, -7 and -3) and slightly higher levels of anti-apoptotic proteins (BCL2, MCL1 and BCL-XL), which may explain this apoptosis resistance. Conclusions: Mutations in ZMYM3 are mainly loss-of-function, associate with NOTCH1 signaling mutations and shorten TFT in CLL patients, suggesting that ZMYM3 mutational status may be a useful marker in the management of early stage CLL patients. Moreover, ZMYM3 mutations reduce histone H4 acetylation and cooperate with NOTCH1 mutations to dysregulate gene-expression, leading to impair DNA damage response and apoptosis evasion.

Valine promotes milk synthesis by regulating PKM2 nuclear accumulation and histone H3 acetylation through the TAS1R1-mTOR-DDX39B signaling pathway

Valine, a branched-chain amino acid found in dairy cows, has been recognized for its critical role in milk synthesis. However, the precise effect of valine on lactation in dairy cows remains an area of investigation. In our study, bovine mammary epithelial cells (BMECs) were isolated to explore the mechanism through which valine enhances milk synthesis. The results showed that 100 μM valine significantly boosted the milk synthesis via TAS1R1-mTOR-DDX39B signaling pathway in BMECs. Subsequent investigations revealed that DDX39B governs the accumulation of PKM2 in the nuclei of BMECs. This nuclear buildup of PKM2 weakened the interaction between HDAC3 and histone H3, leading to an increase in the acetylation levels of histone H3. In an vivo context, the 0.25 % valine-enriched drinking water notably elevated in the expression of milk protein and fat in these mice. Further examination showed that 0.25 % valine drinking water considerably augmented the protein expression levels of DDX39B, PKM2, and p-mTOR in the mice mammary glands. In summary, our results suggest that valine, by modulating the TAS1R1-mTOR-DDX39B signaling pathway, directs the accumulation of PKM2 in the nucleus. This, in turn, escalates the acetylation levels of histone H3, promoting the synthesis of both milk protein and fat.

Unravelling blood-based epigenetic mechanisms: the impact of hsa-miR-146a and histone H3 acetylation in lead-induced inflammation among occupational workers.

Occupational and environmental exposure to lead (Pb) is a persistent health problem majorly in developing countries and has been implied to cause epigenetic alterations. Its effect on histone post-translational modifications is not explored in human population. MicroRNAs are epigenetic modulators reported to be differentially expressed under Pb exposure. The present study was targeted to find plausible association between the role of hsa-miR-146a and global histone (H3) acetylation in Pb-induced inflammation in occupationally exposed workers. A total of 100 occupationally exposed individuals working in different industries were recruited for the study and divided into 2 groups based on the median Pb levels [low Pb group (Pb < 5μg/dL) and High Pb group (Pb > 5μg/dL)]. The Pb levels were measured in whole blood using atomic absorption spectrometry to confirm Pb exposure. Histone H3 acetylation and serum interleukin-6 (IL-6) levels were measured using colorimetric methods and enzyme-linked immunosorbent assay (ELISA), respectively. MicroRNA-146a expression was quantified using TaqMan assay. The median BLL of the study population was 5μg/dL. BLL, IL-6, and Histone (H3) acetylation increased significantly with the duration of exposure. BLL level showed a significant positive correlation with IL-6 and histone H3 acetylation level. We also found that hsa-miR-146a exhibited significantly increased expression in the high Pb group compared to the low Pb group (Fold change: 2.56; P = 0.014). The linear regression model suggested that BLL has significantly predicted histone H3 acetylation, hsa-miR-146a, and IL-6 in the study subjects. The finding that hsa-miR146a was significantly upregulated in individuals with high BLL and had a significant negative correlation with serum IL-6 suggests that Pb-induced oxidative stress likely activates H3 acetylation, which then releases inflammatory cytokines like IL-6.

Sucrose-nonfermenting 1 kinase activates histone acetylase GCN5 to promote cellulase production in Trichoderma.

Trichoderma serves as the primary producer of cellulases and hemicellulases in industrial settings as it readily secretes a variety of cellulolytic enzymes. The protein kinase SNF1 (sucrose-nonfermenting 1) can enable cells to adapt to changes in carbon metabolism by phosphorylating key rate-limiting enzymes involved in the maintenance of energy homeostasis and carbon metabolism within cells. Histone acetylation is an important epigenetic regulatory mechanism that influences physiological and biochemical processes. GCN5 is a representative histone acetylase involved in promoter chromatin remodeling and associated transcriptional activation. Here, the TvSNF1 and TvGCN5 genes were identified in Trichoderma viride Tv-1511, which exhibits promising activity with respect to its ability to produce cellulolytic enzymes for biological transformation. The SNF1-mediated activation of the histone acetyltransferase GCN5 was herein found to promote cellulase production in T. viride Tv-1511 via facilitating changes in histone acetylation. These results demonstrated that cellulolytic enzyme activity and the expression of genes encoding cellulases and transcriptional activators were clearly enhanced in T. viride Tv-1511 mutants in which TvSNF1 and TvGCN5 were overexpressed, with concomitant changes in histone H3 acetylation levels associated with these genes. GCN5 was also found to be directly recruited to promoter regions to alter histone acetylation, while SNF1 functioned upstream as a transcriptional activator that promotes GCN5 upregulation at the mRNA and protein levels in the context of cellulase induction in T. viride Tv-1511. These findings underscore the important role that this SNF1-GCN5 cascade plays in regulating cellulase production in T. viride Tv-1511 by promoting altered histone acetylation, offering a theoretical basis for the optimization of T. viride in the context of industrial cellulolytic enzyme production. KEY POINTS: • SNF1 kinase and GCN5 acetylase promoted cellulase production in Trichoderma by increasing the expression of genes encoding cellulases and transcriptional activators • SNF1 and GCN5 promoted cellulase production by driving H3ac modifications, and GCN5 directly band to the promoter regions to catalyze distinct H3ac modifications • SNF1 acts upstream of GCN5 as a transcriptional activator in the cellulase production of Trichoderma.