Histone Arginine Research Articles

Histone Arginine Methylation as a Regulator of Gene Expression in the Dehydrating African Clawed Frog (Xenopus laevis).

The African clawed frog (Xenopus laevis) endures prolonged periods of dehydration while estivating underground during the dry season. Epigenetic modifications play crucial roles in regulating gene expression in response to environmental changes. The elucidation of epigenetic changes relevant to survival could serve as a basis for further studies on organ preservation under extreme stress. The current study examined the relative protein levels of key enzymes involved in the arginine methylation of histones in the liver and kidney tissues of control versus dehydrated (35 ± 1%) X. laevis through immunoblotting. Protein arginine methyltransferases (PRMT) 4, 5, and 6 showed significant protein level decreases of 35 ± 3%, 71 ± 7%, and 25 ± 5%, respectively, in the liver tissues of the dehydrated frogs relative to controls. In contrast, PRMT7 exhibited an increase of 36 ± 4%. Similarly, the methylated histone markers H3R2m2a, H3R8m2a, and H3R8m2s were downregulated by 34 ± 11%, 15 ± 4%, and 42 ± 12%, respectively, in the livers of dehydrated frogs compared to controls. By contrast, the kidneys of dehydrated frogs showed an upregulation of histone markers. H3R2m2a, H3R8m2a, H3R8m2s, and H4R3m2a were significantly increased by 126 ± 12%, 112 ± 7%, 47 ± 13%, and 13 ± 3%, respectively. These changes can play vital roles in the metabolic reorganization of X. laevis during dehydration, and are likely to increase the chances of survival. In turn, the tissue-specific regulation of the histone arginine methylation mechanism suggests the importance of epigenetic regulation in the adaptation of X. laevis for whole-body dehydration.

Mechanism of Histone Arginine Methylation Dynamic Change in Cellular Stress.

Histone arginine residue methylation is crucial for individual development and gene regulation. However, the dynamics of histone arginine methylation in response to cellular stress remains largely unexplored. In addition, the interplay and regulatory mechanisms between this and other histone modifications are important scientific questions that require further investigation. This study aimed to investigate the changes in histone arginine methylation in response to DNA damage. We report a global decrease in histone H3R26 symmetric dimethylation (H3R26me2s) and hypoacetylation at the H3K27 site in response to DNA damage. Notably, H3R26me2s exhibits a distribution pattern similar to that of H3K27ac across the genome, both of which are antagonistic to H3K27me3. Additionally, histone deacetylase 1 (HDAC1) may be recruited to the H3R26me2s demethylation region to mediate H3K27 deacetylation. These findings suggest crosstalk between H3R26me2s and H3K27ac in regulating gene expression.

Relationship between arginine methylation and vascular calcification

In patients on maintenance hemodialysis (MHD), vascular calcification (VC) is an independent predictor of cardiovascular disease (CVD), which is the primary cause of death in chronic kidney disease (CKD). The main component of VC in CKD is the vascular smooth muscle cells (VSMCs). VC is an ordered, dynamic activity. Under the stresses of oxidative stress and calcium‐‑phosphorus imbalance, VSMCs undergo osteogenic phenotypic transdifferentiation, which promotes the formation of VC. In addition to traditional epigenetics like RNA and DNA control, post-translational modifications have been discovered to be involved in the regulation of VC in recent years. It has been reported that the process of osteoblast differentiation is impacted by catalytic histone or non-histone arginine methylation. Its function in the osteogenic process is comparable to that of VC. Thus, we propose that arginine methylation regulates VC via many signaling pathways, including as NF-B, WNT, AKT/PI3K, TGF-/BMP/SMAD, and IL-6/STAT3. It might also regulate the VC-related calcification regulatory factors, oxidative stress, and endoplasmic reticulum stress. Consequently, we propose that arginine methylation regulates the calcification of the arteries and outline the regulatory mechanisms involved.

Abstract 1740: Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion

Abstract Metastatic potential of basal-like breast cancers typically is initiated by genetic alterations that lead to a process known as epithelial-mesenchymal transition (EMT). However, much is currently not understood regarding the role of epigenetic modifications that lead to invasive characteristics and EMT phenotype of metastatic breast cancers. Based on the previous notion connecting epigenetic changes to breast cancer metastasis, we performed DIA-based mass spectrometry of isolated histones from an isogenic panel of MCF10A breast cell lines where the tumor suppressor genes TP53 and PTEN were silenced and induced EMT. With this approach, we determined which histone modifications were differentially enriched in the non-EMT and EMT-induced MCF10A cell lines. From approximately 72 histone modifications identified and annotated from our mass spectrometry results, we were able to identify 5 histone events that were differentially enriched in our MCF10A cell line panel. Two events of note were histone H3 lysine-14 acetylation (H3K14ac) significantly increasing and histone H4 arginine 55 dimethylation (H4R55me2) significantly decreasing in our EMT-transformed MCF10A p53-/PTEN- cell lines when compared to the parental, non-tumorigenic MCF10A cell line, showing these events are differentially affected during the EMT process in breast cancer cells. Furthermore, significant arginine demethylation of H4R55me2 & H3.1R83me in the EMT-transformed MCF10A p53-/PTEN- cell lines corresponded with JMJD6, an established histone arginine demethylase, being overexpressed in basal-like breast cancer cell lines as well as basal-like breast cancer patients from The Cancer Genome Atlas (TCGA) and METABRIC datasets. Based on histone proteomic profiling of our isogenic cell line model, the loss of specific histone arginine methylation events corresponding with JMJD6 overexpression could highlight the potential for a targetable epigenetic mechanism in breast cancer metastasis. Citation Format: Charlotte B. McGuinness, Megan A. Wilson, Margaret V. Leonard, Maria Ouzounova, Hasan Korkaya, Austin Y. Shull. Histone proteomic profiling of EMT-transformed MCF10A breast cells demonstrates a loss in novel histone arginine methylation sites via dual p53 and PTEN deletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1740.

The In Vitro Effects of Carprofen on Lipopolysaccharide-Induced Neutrophil Extracellular Trap Formation in Dairy Cows.

The objective of this study was to develop an in vitro model that mimics inflammatory reactions and neutrophil extracellular traps (NETs) formation by polymorphonuclear leukocytes (PMNs) in dairy cows. This model was used to examine the effect of carprofen (CA) on lipopolysaccharide (LPS)-induced NETs formation and expression of inflammatory factors. Peripheral blood samples were collected from 24 Holstein cows (3-11 days postpartum) and PMNs were isolated. In three replicates, PMNs were exposed to various treatments to establish an appropriate in vitro model, including 80 μg/mL of LPS for 2 h, followed by co-incubation for 1 h with 60 μmol/L CA and 80 μg/mL LPS. The effects of these treatments were evaluated by assessing NETs formation by extracellular DNA release, gene expression of pro-inflammatory cytokines, reactive oxygen species (ROS) production, and the expression of NETs-related proteins, including histone3 (H3), citrullinated histone (Cit-H3), cathepsin G (CG), and peptidyl arginine deiminase 4 (PAD4). The assessment of these parameters would elucidate the specific mechanism by which CA inhibits the formation of NETs through the PAD4 pathway instead of modulating the Nox2 pathway. This highlights CA's effect on chromatin decondensation during NETs formation. Statistical analyses were performed utilizing one-way ANOVA with Bonferroni correction. The results demonstrated that LPS led to an elevated formation of NETs, while CA mitigated most of these effects, concurrent the PAD4 protein level increased with LPS stimulating and decreased after CA administration. Nevertheless, the intracellular levels of ROS did not change under the presence of LPS. LPS supplementation resulted in an upregulation of H3 and Cit-H3 protein expression levels. Conversely, the CA administration inhibited their expression. Additionally, there was no change in the expression of CG with either LPS or LPS + CA co-stimulation. The gene expression of pro-inflammatory cytokines (tumor necrosis factor -α, interleukin (IL)-18, IL-1β, and IL-6) upregulated with LPS stimulation, while the treatment with CA inhibited this phenomenon. In conclusion, CA demonstrated a pronounced inhibitory effect on both LPS-induced NETs formation as well as the associated inflammatory response.

PRMT5-mediated homologous recombination repair is essential to maintain genomic integrity of neural progenitor cells

Maintaining genomic stability is a prerequisite for proliferating NPCs to ensure genetic fidelity. Though histone arginine methylation has been shown to play important roles in safeguarding genomic stability, the underlying mechanism during brain development is not fully understood. Protein arginine N-methyltransferase 5 (PRMT5) is a type II protein arginine methyltransferase that plays a role in transcriptional regulation. Here, we identify PRMT5 as a key regulator of DNA repair in response to double-strand breaks (DSBs) during NPC proliferation. Prmt5F/F; Emx1-Cre (cKO-Emx1) mice show a distinctive microcephaly phenotype, with partial loss of the dorsal medial cerebral cortex and complete loss of the corpus callosum and hippocampus. This phenotype is resulted from DSBs accumulation in the medial dorsal cortex followed by cell apoptosis. Both RNA sequencing and in vitro DNA repair analyses reveal that PRMT5 is required for DNA homologous recombination (HR) repair. PRMT5 specifically catalyzes H3R2me2s in proliferating NPCs in the developing mouse brain to enhance HR-related gene expression during DNA repair. Finally, overexpression of BRCA1 significantly rescues DSBs accumulation and cell apoptosis in PRMT5-deficient NSCs. Taken together, our results show that PRMT5 maintains genomic stability by regulating histone arginine methylation in proliferating NPCs.

Peptidyl Arginine Deiminase (PAD) 4 Modulates the GI Microbiome and Baseline Inflammation in Mice: Implications for Sepsis

Introduction: As part of the innate immune response, neutrophils release neutrophil extracellular traps (NETs), webs of decondensed chromatin coated with histones that capture bacteria but cause collateral tissue damage when produced in a dysregulated manner. Deficiency of PAD4, a protein that converts histone arginine to citrulline to promote chromatin decondensation essential to NET release (NETosis), has been associated with improved outcomes in murine sepsis models, leading some to propose that inhibiting NETosis may be protective. This conclusion has primarily been drawn from studies using the murine cecal-ligation and puncture (CLP) model of polymicrobial sepsis, in which the cecum is perforated to disseminate GI microbiota. However, NET release is an evolutionarily conserved function, present in protozoa and retained in all studied animal species, suggesting that it plays a crucial role in the immune response. In this study, we seek to characterize the baseline immunophenotype of NET-deficient, PAD4 knockout (PAD4 -/-) mice, to determine how PAD4 influences the GI microbiome to impact outcomes in GI models of polymicrobial sepsis. Methods: Whole blood samples were collected from wild type (WT) C57BL/6 mice, PAD4 -/- mice generated by our group through deletion of exon II in the PAD4 gene (PAD4D ΔII), and PAD4 -/- mice with deletion of exon IX and X (PAD4D ΔIX+X) obtained from Jackson laboratory. Complete blood counts (CBCs) and weights were obtained from PAD4 -/- and healthy WT mice at 9-12 weeks. The Olink target 96 mouse exploratory panel was used to quantify variation in levels of 96 plasma proteins. Next generation sequencing was used to assess the abundance of different bacterial species in stool samples from WT, PAD4D ΔII, and PAD4D ΔIX+X animals. Polymicrobial sepsis was then induced through peritoneal injections of cecal slurry (CS), using cecal samples from WT, PAD4D ΔII, and PAD4D ΔIX+X donor mice. 24 hours post injection, mean sepsis score (MSS) was assessed, and blood was obtained for CBC measurement, plasma proteomic analysis, and quantification of bacterial colony forming units (CFUs). Relative abundance of bacterial species was quantified in blood and liver homogenates. Survival outcome was performed on parallel subsets of WT and PAD4 -/- mice. Results: At comparable ages, both strains of PAD4 -/- animals had lower weights and significantly higher baseline neutrophil counts than WT controls bred in the same colony. Proteomic analysis revealed that PAD4D ΔII mice had baseline elevation in the inflammatory markers IL6 and Plin1, while PAD4D ΔIX+X mice, the strain with more pronounced neutrophilia, had higher GM-CSF and decreased IL23r, proteins crucial to neutrophil mobilization. In CS sepsis studies, WT animals that received WT CS were sicker with higher MSS, lower platelet counts, higher inflammatory markers, and a 30% mortality rate. In contrast, PAD4D ΔII mice that received PAD4D ΔII CS had a milder clinical course, lower bacterial dissemination, and 100% survival (Fig2). To assess the basis for this unexpected outcome, we compared bacterial colony-forming units (CFU) in WT to PAD4 -/- CS and found lower levels of bacteria in PAD4D ΔII CS. Deep sequencing of stool from WT, PAD4D ΔII, and PAD4D ΔIX+X animals revealed marked differences in the relative abundance of bacterial species. Sequencing of blood and liver samples obtained 24 hrs after CS injection showed that the predominant proliferating organism in WT mice was Enterococcaceae species while there was an abundance of Lactobacillaceae species in the blood and liver of PAD4D ΔII mice (Fig1). Sepsis studies repeated with genotype switched CS showed that WT mice treated with PAD4D ΔIX+X CS had a milder disease course with 100% survival, whereas PAD4D ΔIX+X mice injected with WT CS had elevated MSS, a trend towards higher plasma inflammatory markers, and a 50% mortality rate, similar to that observed in WT mice injected with WT CS (Fig2). Conclusions: These studies show that NETosis influences baseline inflammation and modulates the GI microbiome in mice. Differences in disease severity and survival between PAD4 -/- and WT mice observed in the CS model of polymicrobial sepsis is primarily due to greater pathogenicity of the WT CS inoculum rather than collateral tissue damage secondary to dysregulated NETosis. The basis for differences in inflammation and microbiome composition in PAD4 -/- mice needs to be examined.

Natural products modulating epigenetic mechanisms by affecting histone methylation/demethylation: targeting cancer cells.

Many natural products can exert anticancer or chemopreventive activity by interfering with the cellular epigenetic machinery. While a plethora of studies indicate the relevance of affecting DNA methylation and histone acetylation, the influence on the mechanisms related to histone methylation is often overlooked. This may be associated with the lagging evidence that changes in the action of histone methylation writers and erasers and subsequent alterations in the profile of histone methylation are causally related with carcinogenesis. However, recent animal studies have shown that targeting histone methylation/demethylation affects the course of experimentally induced carcinogenesis. Existing data suggest that numerous natural compounds from different chemical groups, including green tea polyphenols and other flavonoids, curcuminoids, stilbene derivatives, phenolic acids, isothiocyanates, alkaloids and terpenes, can affect the expression and activity of crucial enzymes involved in the methylation and demethylation of histone lysine and arginine residues. These activities have been associated with the modulation of cancer-related gene expression and functional changes, including reduced cell proliferation and migration, and enhanced apoptosis in various cancer models. Most studies focused on the modulation of the expression and/or activity of two proteins - EZH2 (a H3K27 methyltransferase) and LSD1 (a H3K4/9 demethylase), or the effects on the global levels of histone methylation marks by the phytochemicals, but data regarding other histone methyltransferases or demethylases are scarce. While the field still remains relatively unexplored, this review aims to explore the impact of natural products on the enzymes related to histone methylation/demethylation, showing their relevance to carcinogenesis and cancer progression.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial–temporal regulation of histone arginine methylation in neurodevelopment

BackgroundOxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency.ResultsWe report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial–temporal regulation of histone arginine methylation in specific brain regions.ConclusionsThis study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

CARM1 deficiency inhibits osteoblastic differentiation of bone marrow mesenchymal stem cells and delays osteogenesis in mice

Bone repair remains a clinical challenge due to low osteogenic capacity. Coactivator associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that mediates arginine methylation and endochondral ossification. However, the roles of CARM1 in osteoblastic differentiation and bone remodeling have not been explored. In our study, heterozygous CARM1-knockout (KO) mice were generated using the CRISPR-Cas9 system and a model of femoral defect was created. At day 7 postsurgery, CARM1-KO mice exhibited obvious bone loss compared with wild type (WT) mice, as evidenced by reduced bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Deletion of CARM1 in mice lowered synthesis and accumulation of collagen at the injury sites. The alkaline phosphatase (ALP) activity and osteogenic-related gene expression were declined in CARM1-KO mice. To further understand the role of CARM1 in osteoblastic differentiation, bone marrow mesenchymal stem cells (BMSCs) were isolated from the tibia and femur of WT or CARM1-KO mice. CARM1 deletion decreased histone arginine methylation and inhibited osteoblastic differentiation and mineralization. The mRNA sequencing of CARM1-KO BMSCs revealed the possible regulatory molecules by CARM1, which could deepen our understanding of CARM1 regulatory mechanisms. These data could be of interest to basic researchers and provide the direction for future research into bone-related disorders.

Role of histone methylation in skin cancers: Histone methylation-modifying enzymes as a new class of targets for skin cancer treatment.

Histone methylation, one of the most prominent epigenetic modifications, plays a vital role in gene transcription, and aberrant histone methylation levels cause tumorigenesis. Histone methylation is a reversible enzyme-dependent reaction, and histone methyltransferases and demethylases are involved in this reaction. This review addresses the biological and clinical relevance of these histone methylation-modifying enzymes for skin cancer. In particular, the roles of histone lysine methyltransferases, histone arginine methyltransferase, lysine-specific demethylases, and JmjC demethylases in skin cancer are discussed in detail. In addition, we summarize the efficacy of several epigenetic inhibitors targeting histone methylation-modifying enzymes in cutaneous cancers, such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma. In conclusion, we propose histone methylation-modifying enzymes as novel targets for next-generation pharmaceuticals in the treatment of skin cancers and further provide a rationale for the development of epigenetic drugs (epidrugs) that target specific histone methylases/demethylases in cutaneous tumors.

JMJD6 protects against isoproterenol-induced cardiac hypertrophy via inhibition of NF-κB activation by demethylating R149 of the p65 subunit.

Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. In this study we investigated the role of a histone arginine demethylase, Jumonji C domain-containing protein 6 (JMJD6) in pathological cardiac hypertrophy. Cardiac hypertrophy was induced in rats by subcutaneous injection of isoproterenol (ISO, 1.2 mg·kg-1·d-1) for a week. At the end of the experiment, the rats underwent echocardiography, followed by euthanasia and heart collection. We found that JMJD6 levels were compensatorily increased in ISO-induced hypertrophic cardiac tissues, but reduced in patients with heart failure with reduced ejection fraction (HFrEF). Furthermore, we demonstrated that JMJD6 overexpression significantly attenuated ISO-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs) evidenced by the decreased cardiomyocyte surface area and hypertrophic genes expression. Cardiac-specific JMJD6 overexpression in rats protected the hearts against ISO-induced cardiac hypertrophy and fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) exacerbated ISO-induced hypertrophic responses in NRCMs. We revealed that JMJD6 interacted with NF-κB p65 in cytoplasm and reduced nuclear levels of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 bound to p65 and demethylated p65 at the R149 residue to inhibit the nuclear translocation of p65, thus inactivating NF-κB signaling and protecting against pathological cardiac hypertrophy. In addition, we found that JMJD6 demethylated histone H3R8, which might be a new histone substrate of JMJD6. These results suggest that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome

Epigenetic mechanisms oversee epidermal homeostasis and oncogenesis. The identification of kinases controlling these processes has direct therapeutic implications. We show that ULK3 is a nuclear kinase with elevated expression levels in squamous cell carcinomas (SCCs) arising in multiple body sites, including skin and Head/Neck. ULK3 loss by gene silencing or deletion reduces proliferation and clonogenicity of human keratinocytes and SCC-derived cells and affects transcription impinging on stem cell-related and metabolism programs. Mechanistically, ULK3 directly binds and regulates the activity of two histone arginine methyltransferases, PRMT1 and PRMT5 (PRMT1/5), with ULK3 loss compromising PRMT1/5 chromatin association to specific genes and overall methylation of histone H4, a shared target of these enzymes. These findings are of translational significance, as downmodulating ULK3 by RNA interference or locked antisense nucleic acids (LNAs) blunts the proliferation and tumorigenic potential of SCC cells and promotes differentiation in two orthotopic models of skin cancer.

Mechanistic aspects of reversible methylation modifications of arginine and lysine of nuclear histones and their roles in human colon cancer.

Developmental proceedings and maintenance of cellular homeostasis are regulated by the precise orchestration of a series of epigenetic events that eventually control gene expression. DNA methylation and post-translational modifications (PTMs) of histones are well-characterized epigenetic events responsible for fine-tuning gene expression. PTMs of histones bear molecular logic of gene expression at chromosomal territory and have become a fascinating field of epigenetics. Nowadays, reversible methylation on histone arginine and lysine is gaining increasing attention as a significant PTM related to reorganizing local nucleosomal structure, chromatin dynamics, and transcriptional regulation. It is now well-accepted and reported that histone marks play crucial roles in colon cancer initiation and progression by encouraging abnormal epigenomic reprogramming. It is becoming increasingly clear that multiple PTM marks at the N-terminal tails of the core histones cross-talk with one another to intricately regulate DNA-templated biological processes such as replication, transcription, recombination, and damage repair in several malignancies, including colon cancer. These functional cross-talks provide an additional layer of message, which spatiotemporally fine-tunes the overall gene expression regulation. Nowadays, it is evident that several PTMs instigate colon cancer development. How colon cancer-specific PTM patterns or codes are generated and how they affect downstream molecular events are uncovered to some extent. Future studies would address more about epigenetic communication, and the relationship between histone modification marks to define cellular functions in depth. This chapter will comprehensively highlight the importance of histone arginine and lysine-based methylation modifications and their functional cross-talk with other histone marks from the perspective of colon cancer development.

Histone arginine methylation in the freeze-tolerant wood frog, Rana sylvatica

Histone arginine methylation in the freeze-tolerant wood frog, Rana sylvatica

METTL23 mutation alters histone H3R17 methylation in normal-tension glaucoma.

Normal-tension glaucoma (NTG) is a heterogeneous disease characterized by retinal ganglion cell (RGC) death leading to cupping of the optic nerve head and visual field loss at normal intraocular pressure (IOP). The pathogenesis of NTG remains unclear. Here, we describe a single nucleotide mutation in exon 2 of the methyltransferase-like 23 (METTL23) gene identified in 3 generations of a Japanese family with NTG. This mutation caused METTL23 mRNA aberrant splicing, which abolished normal protein production and altered subcellular localization. Mettl23-knock-in (Mettl23+/G and Mettl23G/G) and -knockout (Mettl23+/- and Mettl23-/-) mice developed a glaucoma phenotype without elevated IOP. METTL23 is a histone arginine methyltransferase expressed in murine and macaque RGCs. However, the novel mutation reduced METTL23 expression in RGCs of Mettl23G/G mice, which recapitulated both clinical and biological phenotypes. Moreover, our findings demonstrated that METTL23 catalyzed the dimethylation of H3R17 in the retina and was required for the transcription of pS2, an estrogen receptor α target gene that was critical for RGC homeostasis through the negative regulation of NF-κB-mediated TNF-α and IL-1β feedback. These findings suggest an etiologic role of METTL23 in NTG with tissue-specific pathology.

Epigenetics in glaucoma: a link between histone methylation and neurodegeneration

Normal-tension glaucoma is a form of optic nerve degeneration that is characterized by loss of retinal ganglion cells independent of eye pressure elevation. In this issue of the JCI, Pan et al. report the discovery in a Japanese family of a mutation in the METTL23 gene, which encodes a histone arginine methyltransferase that causes normal-pressure glaucoma in haploinsufficiency. Inherited as an autosomal dominant condition, METTL23 deficiency revealed an important function in the regulation of pS2 and the downstream NF-κB signaling pathway, which has previously been linked to glaucomatous optic nerve degeneration. These findings are the first direct link between defective epigenetic regulatory machinery and genetic forms of optic nerve degeneration.

Loss of PRMT7 reprograms glycine metabolism to selectively eradicate leukemia stem cells in CML

Our group has reported previously on the role of various members of the protein arginine methyltransferase (PRMT) family, which are involved in epigenetic regulation, in the progression of leukemia. Here, we explored the role of PRMT7, given its unique function within the PRMT family, in the maintenance of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Genetic loss of Prmt7, and the development and testing of a small-molecule specific inhibitor of PRMT7, showed that targeting PRMT7 delayed leukemia development and impaired self-renewal of LSCs in a CML mouse model and in primary CML CD34+ cells from humans without affecting normal hematopoiesis. Mechanistically, loss of PRMT7 resulted in reduced expressions of glycine decarboxylase, leading to the reprograming of glycine metabolism to generate methylglyoxal, which is detrimental to LSCs. These findings link histone arginine methylation with glycine metabolism, while suggesting PRMT7 as a potential therapeutic target for the eradication of LSCs in CML.

Inhibition of PRMT5 Disrupts Cell Cycle Progression and DNA Damage Signaling, Revealing a Potential Novel Combination Therapy for Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and remains a therapeutic challenge. There is an urgent need for novel therapeutic strategies for PDAC, and the targeting of pathways highly relevant to its pathobiology is critical. PDAC cells often possess defects in DNA damage repair (DDR) pathways that are not present in healthy cells, which can be exploited for therapeutic strategies. Protein arginine methyltransferase 5 (PRMT5) is a type II histone arginine methyltransferase involved in various cellular processes, such as cell cycle progression and DDR. In PDAC tumors, PRMT5 expression is elevated and predicts shorter overall survival and disease‐free patient survival than those with lower PRMT5 expression. Here, we examined the effect of PRMT5 inhibition on PDAC in vitro, the impact of its inhibition on cell cycle and DDR, as well as potential synergy between PRMT5 inhibition and DNA damage‐inducing ionizing radiation (IR) as a novel therapeutic strategy for PDAC. Treatment of PDAC cells withthe PRMT5 inhibitor (PRMT5i), EPZ015938, resulted in a dose‐dependent decrease of cell growth by time‐lapse live‐cell imaging and clonogenic survival assays. Flow cytometry‐based cell cycle analysis showed that PDAC cells treated with EPZ015938 had increased numbers in G2/M, suggesting that PRMT5 inhibition triggers a G2/M arrest. Using a phospho‐protein array of cell cycle control proteins, we found higher levels of WEE1, P‐S216‐CDC25C, and P‐Y15‐CDC2, supporting G2/M arrest following treatment with EPZ015938. The phospho‐protein array also revealed an increase in P‐S15‐TP53, a signal of DNA damage mediated by the DNA damage sensors, ATM/ATR, along with a decrease in ATM. We confirmed the ATM downregulation at the transcript level by quantitative RT‐PCR. Pathway‐specific qPCR array analysis displayed differential expression of DDR genes related to ATM and G2/M DNA damage checkpoint regulation, further implicating a mechanistic role for these pathways in response to PRMT5i. The downregulation of ATM combined with the presence of G2/M arrest and signals of DNA damage led us to examine the related ATR pathway. In protein lysates from PDAC cells treated with PRMT5i followed by western blot, we found increased phosphorylation of ATR at S1981 and H2A.X at S139. This suggests that PRMT5 inhibition activates DDR in PDAC cells via ATR. To harness the potential vulnerability presented by PRMT5i‐mediated G2/M arrest, we combined PRMT5i treatment with ionizing radiation (IR) exposure to find that the combination of EPZ015938 and IR displayed a greater reduction in PDAC cell growth in vitro over individual treatments. In conclusion, we find that PRMT5 is a critical player in DNA damage via modulation of the ATM/ATR pathways, and combined PRMT5i and IR have beneficial anti‐tumor effects in vitro, revealing an exciting avenue for further exploration as a potential new therapeutic strategy in PDAC.

Novel Chemicals Derived from Tadalafil Exhibit PRMT5 Inhibition and Promising Activities against Breast Cancer.

Breast cancer seriously endangers women’s health worldwide. Protein arginine methyltransferase 5 (PRMT5) is highly expressed in breast cancer and represents a potential druggable target for breast cancer treatment. However, because the currently available clinical PRMT5 inhibitors are relatively limited, there is an urgent need to develop new PRMT5 inhibitors. Our team previously found that the FDA-approved drug tadalafil can act as a PRMT5 inhibitor and enhance the sensitivity of breast cancer patients to doxorubicin treatment. To further improve the binding specificity of tadalafil to PRMT5, we chemically modified tadalafil, and designed three compounds, A, B, and C, based on the PRMT5 protein structure. These three compounds could bind to PRMT5 through different binding modes and inhibit histone arginine methylation. They arrested the proliferation and triggered the apoptosis of breast cancer cells in vitro and also promoted the antitumor effects of the chemotherapy drugs cisplatin, doxorubicin, and olaparib in combination regimens. Among them, compound A possessed the highest potency. Finally, the anti-breast cancer effects of PRMT5 inhibitor A and its ability to enhance chemosensitivity were further verified in a xenograft mouse model. These results indicate that the new PRMT5 inhibitors A, B, and C may be potential candidates for breast cancer treatment.