Protein Methylation Research Articles

ALKBH5 protects against stroke by reducing endoplasmic reticulum stress-dependent inflammation injury via the STAT5/PERK/EIF2α/CHOP signaling pathway in an m6A-YTHDF1-dependent manner

BackgroundEndoplasmic reticulum (ER) stress causes neuroinflammation and neuronal apoptosis during ischemic stroke progression. This study has investigated the role of ALKBH5 in ER stress during ischemic stroke progression. MethodsIn vivo and in vitro models of ischemic stroke were established by middle cerebral artery occlusion (MCAO) and OGD/R treatment, respectively. Cerebral infarct size was detected using triphenyltetrazolium chloride staining (TTC), and pathological changes were examined using histological staining. The levels of inflammatory factors were analyzed using Enzyme-linked immunosorbent assay. Cell counting kit-8 assay and flow cytometry were used to measure cell viability and apoptosis, respectively. The global m6A level was detected using the commercial kit, and STAT5 mRNA m6A level was determined using methylated RNA binding protein immunoprecipitation (Me-RIP). ALKBH5, YTHDF1, and STAT5 interactions were analyzed using RIP and RNA pull-down assays. ResultsALKBH5 was upregulated in MCAO animals and OGD/R cell models. ALKBH5 knockdown exacerbated ER stress, neuroinflammation, and neuronal apoptosis in brain tissues and neuronal cells. ALKBH5 inhibited STAT5 mRNA stability and expression in an m6A-YTHDF1-dependent manner. STAT5 promoted ER stress by activating the PERK/eIF2/CHOP signaling pathway. Furthermore, STAT5 knockdown reversed the effects of ALKBH5 knockdown on OGD/R-induced ER stress and neuroinflammation in HT22 cells. ConclusionALKBH5 knockdown exacerbated ischemic stroke by increasing ER stress-dependent neuroinflammation and neuronal apoptosis via the STAT5/PERK/EIF2α/CHOP signaling pathway in an m6A-YTHDF1-dependent manner.

RNA N6-methyladenosine-modified-binding protein YTHDF1 promotes prostate cancer progression by regulating androgen function-related gene TRIM68

PurposeThere is no report about the direct relationship between m6A modification and androgen receptor (AR)-related genes in prostate cancer (PC). We aimed to study the mechanisms of m6A methylation in regulating the pathogenesis of PC from the perspective of AR-related genes.MethodsqRT-PCR was applied to detect the expression of m6A-related genes in PC cell with or without AR inhibitor. The effects of YTHDF1 knockdown on PC cell viability, apoptosis, migration and invasion were investigated using flow cytometry, wound healing and transwell assays, respectively. The mechanism of YTHDF1 action was investigated using m6A RNA immunoprecipitation (MeRIP) sequencing. The biological functions of YTHDF1 were also explored through in vivo experiments.ResultsYTHDF1 was significantly down-regulated in AR inhibitor group. YTHDF1 knockdown significantly decreased AR level, viability and m6A methylation level of PC cells. TRIM68 was identified as a direct target of YTHDF1. Both YTHDF1 and TRIM68 knockdown increased apoptosis, and decreased cell viability, migration, and invasion of PC cells, while TRIM68 overexpression reversed the effects of YTHDF1 knockdown on PC cells. In addition, knockdown of YTHDF1 or TRIM68 significantly decreased the m6A methylation level, and mRNA and protein levels of YTHDF1, TRIM68 and AR in PC cells, while TRIM68 overexpression increased the expression levels above. Furthermore, subcutaneous xenografts of nude mice also revealed that TRIM68 could reverse the effects of YTHDF1 knockdown in PC in vivo.ConclusionThis study suggested the key role of YTHDF1-mediated m6A modification in PC progression by regulating androgen function-related gene TRIM68 in PC.

Abstract PR006: Initial results from first-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in biomarker-selected solid tumors

Abstract Background: AMG 193 is a second-generation protein arginine methyltransferase 5 (PRMT5) inhibitor that targets the MTA-bound state of PRMT5 in methylthioadenosine phosphorylase (MTAP)-null tumors. PRMT5 is responsible for methylation and gene silencing of cell-essential proteins dysregulated in cancer and is partially inhibited in tumors harboring MTAP deletion, which occurs in ~15% of solid tumors. First generation PRMT5 inhibitors were intolerable due to indiscriminate inhibition of PRMT5 leading to dose-limiting myelosuppression. In preclinical studies, AMG 193 demonstrated selective antitumor activity in MTAP-null models by further suppressing PRMT5 function while sparing normal function, thereby improving upon first generation molecules. We report the initial clinical results from dose-escalation in the ongoing first-in-human (FIH) study. Methods: AMG193 was orally administered in continuous 28-day cycles to patients (pts) with advanced MTAP-null solid tumors. Dose escalation proceeded via a BLRM method. The primary objectives include safety, tolerability, and identification of the maximum tolerated dose (MTD). Secondary objectives include preliminary antitumor activity by investigator-assessed RECIST, pharmacokinetics (PK) and pharmacodynamic (PD) effects. Results: As of August 8, 2023, 47 pts with MTAP-null cancer (PDAC n = 10; NSCLC n = 6; CCA = 5; MESO n = 3; others n = 23) were enrolled in seven escalating cohorts. Five pts had DLTs, and exploration continues per protocol to identify the MTD. The most common TRAEs were nausea (45%), fatigue (26%), decreased appetite (17%), and vomiting (17%). Preliminary PK analyses showed dose-proportional systemic exposure with a half-life of 7–11 hrs. Among 31 pts who had at least one postbaseline scan, there were 5 with confirmed PRs [PDAC (–100%), ovarian Sertoli-Leydig (–59%), RCC (–58%), esophageal (–46%), and gallbladder cancer (–63%), 1 each], 14 with stable disease (including 9 with some degree of tumor shrinkage), and 12 with disease progression. All PRs were ongoing at the data cutoff with treatment durations of 140–275 days. PD effects demonstrated dose-dependent reduction in serum total SDMA levels and complete PRMT5 inhibition was confirmed in five pts with on-treatment biopsies spanning multiple dose levels. Exploratory analysis of changes in variant allele frequency by ctDNA demonstrated rapid treatment effects that was predictive and correlated with response. Conclusion: AMG 193 is an MTA-cooperative PRMT5 inhibitor designed to induce synthetic lethality in MTAP-null solid tumors while sparing hematologic toxicity. The initial results of the FIH study demonstrate proof-of-concept with encouraging signs of preliminary clinical activity without evidence of myelosuppression. Dose escalation continues to proceed to establish the MTD. AMG 193 has demonstrated promise as a potential new therapeutic for pts with tumors that have MTAP loss. Citation Format: Jordi Rodon, Noboru Yamamoto, Toshihiko Doi, François Ghiringhelli, Maria-Elisabeth Goebeler, Yutaka Fujuwara, Miguel Villalona-Calero, Nicolas Penel, Amita Patnaik, Jean-Pascal Machiels, Alfredo Addeo, James Abbruzzese, Sophie Postel-Vinay, Sylvie Rottey, Chunxu Liu, Chen-Hua Chuang, William Kormany, Paul Hughes, Kiana Keyvanjah, Bert O'Neil. Initial results from first-in-human study of AMG 193, an MTA-cooperative PRMT5 inhibitor, in biomarker-selected solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR006.

Dietary supplementation of pomegranate extract alters the Caspase-3 and VEGF promoter epigenetic methylation status and protein levels in breast cancer patients undergoing chemotherapy

Dietary supplementation of pomegranate extract alters the Caspase-3 and VEGF promoter epigenetic methylation status and protein levels in breast cancer patients undergoing chemotherapy

PRMT1 Promotes Chronic Lymphocytic Leukemia Progression Via Modulating Methylation of MAST1

Introduction As a member of the protein arginine N-methyltransferase family, protein arginine methyltransferase 1 (PRMT1) plays a crucial regulatory role in the etiology of malignant neoplasms. PRMT1-mediated protein methylation promoted the maintenance of acute myeloid leukemia, which revealed the promising potency of PRMT1 inhibitors. Recent study suggested that dysregulation of PRMT5 promotes Richter's transformation in chronic lymphocytic leukemia (CLL). However, the effects of PRMT1 in the tumorigenesis and progression of CLL still remained ill-defined. Hence, the aim of this study was to investigate the clinical significance and mechanisms of PRMT1 underlying the development of CLL. Methods Peripheral blood samples were collected from 79 newly diagnosed CLL patients (47 males and 32 females; age range 39-85 years, median 63 years) in Shandong Provincial Hospital CLL (SPHCLL) cohort with informed consent. CRISPR-Cas9 technology was used to stably knockout PRMT1 in CLL cells. A label free quantitative proteomics analysis was implemented to reveal the protein methylation mediated by PRMT1. Assessment of cell viability, apoptosis and cell cycle were analyzed by cell counting kit-8, annexin V-PE/7AAD and PI/ RNase staining, respectively. This study was approved by the Medical Ethics Committee of Shandong Provincial Hospital. Results This study examined the expression of PRMT1 in SPHCLL and GEO databases, and discovered the upregulation of PRMT1 mRNA in CLL cells. Aberrantly elevated expression of PRMT1 was observed in a cohort of newly diagnosed CLL patients than healthy donors in mRNA level (donors vs. CLL patients, 0.07±0.06 vs. 0.20 ±0.21, p=0.013). Furthermore, increased expression of PRMT1 was correlated with inferior prognosis in two long-term follow-up cohorts of CLL patients (HR=2.80, p=0.002, and HR=1.551, p=0.009). To elucidate the functional significance of PRMT1 in CLL, we established stable PRMT1 knockdown cells using lentiviral shRNAs and PRMT1 knockout cells using CRISPR/Cas9 technology. The suppression of PRMT1 remarkably inhibited cell proliferation, induced cell apoptosis and blocked cell cycle at G1/S phase in CLL cells. Furthermore, it was observed that C7280948, a selective inhibitor of PRMT1, resulted in the defective proliferation of CLL primary cells in a dose-dependent manner. We subsequently performed pre-clinical investigations of C7280948 in the orthotopic CLL xenograft murine model. Notably, the administration of C7280948 significantly diminished the leukemia burden and induced the lessened degree of splenomegaly in comparison to the control group (Fig. 1A). Moreover, a reduction in the population of CLL cells was detected in bone marrow and spleen of mice treated with C7280948. To decipher the role of PRMT1 in the pathogenesis of CLL, the label free quantitative proteomics analysis was conducted on PRMT1-deficent and control MEC1 cells. Knockdown of PRMT1 diminished asymmetric dimethylarginine of some proteins in CLL cells. In accordance with the results of proteomics analysis, microtubule associated serine/threonine kinase 1 (MAST1) was selected as a candidate target for further investigation. The methylation site of MAST1 protein was identified as R802 (Fig. 1B). Increased expression of MAST1 was correlated with inferior prognosis of CLL patients ( p<0.001), indicating the essential role of MAST1 in the progression of CLL. These results supported the hypothesis that PRMT1 interacted with MAST1 and enhanced the activity of MAST1 via methylating this oncoprotein in CLL cells. Conclusion The present study provides robust evidence for the oncogenic function of PRMT1 in the pathogenesis of CLL, highlighting the potential therapeutic efficacy of the selective PRMT1 inhibitor C7280948. Collectively, the targeted inhibition of PRMT1 exhibits promising prospects for the management of CLL patients.

Inactivation of DNA Polymerase Theta (PolΘ) Is Synthetic Lethal in DNMT3A Mutated Myeloid Malignancies - Potential Clinical Applications

Somatic mutations in DNMT3A are associated with unfavorable outcome in patients with AML, MPN and CML. DNMT3A mutations promote resistance to anthracyclines (including daunorubicin, the component of standard “7+3” induction therapy), interferon alpha, and ABL1 kinase inhibitor imatinib. Thus, malignant clones carrying DNMT3A mutations may be difficult to eliminate using standard treatments. AML, MPN and CML cells harbor oncogenic tyrosine kinase (OTK) such as FLT3(ITD), JAK2(V617F) and BCR-ABL1, respectively. We reported before that elevated levels of formaldehyde generated by altered serine/one-carbon cycle metabolism contributed to accumulation of highly lethal DNA double-strand breaks (DSBs) in OTK-positive cells. To protect malignant cells from DSB-induced apoptosis, OTKs regulate DNA damage response (DDR) mechanisms involving DSBs sensing (ATM and ATR kinases) and repairing (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, Polθ-dependent microhomology-mediated end-joining = TMEJ) as well as these activating cell cycle checkpoints (CHK1 and CHK2 kinases). Unfortunately, DNMT3A mutations caused resistance of OTK-positive cells to numerous DDR inhibitors (DDRis). DDRis sensitivity screen and synthetic lethal CRISPR/Cas9 screen revealed that OTK-positive cells with DNMT3A mutations are uniquely sensitive to the inhibition of DNA polymerase theta (Polθ encoded by POLQ gene). This effect was dependent on generation of formaldehyde by serine/one-carbon cycle metabolism. Abrogation of DNMT3A function by CRISPR/Cas9 targeting, Cre-loxP gene deletion, and heterozygous R882H mutation resulted in hypersensitivity of OTK-positive murine AML-like cells and human primary AML cells to Polθ inhibitors (Polθis) due to accumulation of toxic DSBs and activation of cGAS/STING pro-apoptotic pathway. Moreover, simultaneous loss of functional DNMT3A combined with inactivation of Polθ (by CRISPR/Cas9 targeting, insertion of neo-resistance gene, and D2230A+Y2231A polymerase inactive mutant) caused accumulation of DSBs, and reduced OTK-driven clonogenic potential and leukemogenic activity in mice. Polθ is abundantly overexpressed in OTK-positive DNMT3A-deficient cells due to enhanced POLQ mRNA stability and elevated translation of Polθ protein but not due to altered POLQ methylation and Polθ protein stability. Polθ is a key element not only in TMEJ of DSBs with limited end-resection, but also in replication fork restart and in single-strand DNA (ssDNA) gap filling. OTK-positive DNMT3A-deficient cells displayed hyperactivity of Polθ-mediated TMEJ and replication fork restart, but not ssDNA gap filling. These effects were accompanied by increased loading of Polθ on DNA damage detected by Polθ foci formation and chromatin extraction. Moreover, DNMT3A deficiency modulates chromatin architecture at DSBs to limit DNA end-resection thus favoring TMEJ over HR. Furthermore, we tested the effectiveness of Polθis combined with FDA approved drugs (quizartinib, etoposide, cytarabine, azacytidine) against FLT3(ITD)-positive DNMT3A-deficient cells (primary patient cells and cell lines) in vitro and in vivo. The combination of Polθis + quizartinib and Polθis + etoposide completely eradicated clonogenic activity of these cells while Polθis + cytarabine and Polθis + azacytidine exerted modest and weak effects, respectively, when compared to individual compound treatments. These drug combinations were only modestly toxic to normal bone marrow cells. Treatment with Polθi or etoposide reduced the percentage of GFP+ FLT3(ITD)-positive DNMT3A-deficient leukemia cells in peripheral blood of the mice by ~2-fold and prolonged survival time by ~1.5-fold. Remarkably, the combination of Polθi and etoposide eradicated leukemia cells below detectable levels in 6/12 mice with no visible toxicity. Median survival time of the mice will be recorded. Altogether, we discovered that Polθ protects OTK-positive DNMT3A-deficient myeloid malignant cells from the toxic effects of DSBs and identified Polθ as a novel therapeutic target.

Comparative Transcriptome Analyses in Mantle Cell Lymphoma (MCL): Evidence of E2F1 As a Major Target in Aggressive Blastoid MCL Model

Background Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma characterized by the translocation t(11;14)(q13;q32) which triggers the dysregulation of the cyclin D1 by placing its coding gene CC1ND1 under the control of an immunoglobulin promoter. One of the major consequences of this molecular event is the disturbance of the cyclin D/Rb pathway leading to Rb inactivation and activation the elongation factor E2F1 mainly implicated in cell cycle progression. The mainstay of MCL therapies remains the chemo-immunotherapy followed by autologous transplantation but recently several novel therapies have been developed such as BTK inhibitors, Venetoclax, CDK4/6 inhibitors and more recently CAR-T therapies. However, patients with aggressive forms of MCL exhibit low response rates, requiring the identification of novel therapeutic targets. Materials and Methods We have previously generated an MCL cell line (UPN-1) from a patient with a blastoid variant of MCL presenting unique morphological and cytogenetics characteristics (M'kacher et al, Oncogene 2003). This cell line has been shown to harbor a TP53 mutation and a highly increased sensitivity to radiation despite the absence of functional p53. The molecular basis of this phenomenon has not been explored. In this work, we further characterized UPN-1 cell line using the Clarium S human microarray which has been compared to the gene expression profile of other existing MCL cell lines (GRANTA-519, JEKO-1, JVM-2, MAVEK-1, MINO, REC-1, SP-49, SP-53, Z-138). After mathematical correction, a supervised analysis was performed between triplicate samples of UPN-1 and a group constituted with all other MCL cell lines. We have then set up in vitro assays to determine the effect of a direct E2F1 inhibitor on the proliferative potential of UPN-1. Results The comparative transcriptome analyses revealed a list of 47 genes specifically upregulated in UPN-1. Unsupervised classification allowed to discriminate UPN-1 samples from other MCL cell lines based on the expression of these 47 genes. These data suggested a specific expression profile of UPN-1 as compared to other cell lines. Among the upregulated genes, E2F1 elongation factor was found to be highly upregulated in UPN-1 as compared to other MCL cell lines along with positive enrichment of E2F targets which are mainly implicated in cell cycle progression (Figure 1). Interestingly, UPN-1 has a low level of BCL-2 as compared to other MCL lines that could explain its radio sensitivity. However, other signaling pathways involved in the inhibition of apoptosis were found to be present in UPN-1 by geneset enrichment analysis. The expression of CDKN1A (p21), major cell cycle regulator and known as a marker of aggressive MCL, was found to be low in UPN-1. A repression of immune response pathway such as NFKB with low expression of NFKBIA was identified. Finally, the expression of PIK3CD which encodes for a therapeutic target in MCL (such as PI3KDelta inhibitor Idelalisib) was also significantly repressed in UPN-1 as compared to nine other MCL cell lines. To evaluate the effect of the direct inhibition of E2F1, we treated UPN-1 cells with a targeted E2F1 inhibitor 5‘-Deoxy-5‘-(methylthio)adenosine (MTA) which is a protein methylation inhibitor. MTT assays revealed a 50% reduction of proliferation at + 4 hours in comparison to the vehicle-treated conditions (P<0.0001).Annexin assays showed an increase in early apoptosis (36.6 %) at + 8 hours with subsequent rise in late apoptotic cells (64.5 %) observed after 24 hours of treatment. Interestingly, qRT-PCR analyses of MTA-treated cells exhibited a decrease in the expression of downstream factors associated with E2F1, such as RAD51, BRCA1, CDK2, CCNB2, RANBP1. Conclusions We report here for the first time the comparative transcriptome analysis of the unique aggressive blastoid MCL line UPN-1 as compared to other MCL lines described. This cell line exhibited a major upregulation of E2F1 expression and a down regulation of the cell cycle inhibitor CDKN1A(p21). The potential effects of the direct inhibition of E2F1 using the targeted inhibitor MTA demonstrated a reduction in cell proliferation and induction of apoptosis, suggesting the possibility of associating clinically acceptable E2F1 inhibition strategies to other current therapies.

Targeting protein methylation in pancreatic cancer cells results in KRAS signaling imbalance and inhibition of autophagy

Pancreatic cancer cells with mutant KRAS require strong basal autophagy for viability and growth. Here, we observed that some processes that allow the maintenance of basal autophagy in pancreatic cancer cells are controlled by protein methylation. Thus, by maintaining the methylation status of proteins such as PP2A and MRAS, these cells can sustain their autophagic activity. Protein methylation disruption by a hypomethylating treatment (HMT), which depletes cellular S-adenosylmethionine levels while inducing S-adenosylhomocysteine accumulation, resulted in autophagy inhibition and endoplasmic reticulum stress-induced apoptosis in pancreatic cancer cells. We observed that by reducing the membrane localization of MRAS, hypomethylation conditions produced an imbalance in KRAS signaling, resulting in the partial inactivation of ERK and hyperactivation of the PI3K/AKT–mTORC1 pathway. Interestingly, HMT impeded CRAF activation by disrupting the ternary SHOC2 complex (SHOC2/MRAS/PP1), which functions as a CRAF-S259 holophosphatase. The demethylation events that resulted in PP2A inactivation also favored autophagy inhibition by preventing ULK1 activation while restoring the cytoplasmic retention of the MiT/TFE transcription factors. Since autophagy provides pancreatic cancer cells with metabolic plasticity to cope with various metabolic stress conditions, while at the same time promoting their pathogenesis and resistance to KRAS pathway inhibitors, this hypomethylating treatment could represent a therapeutic opportunity for pancreatic adenocarcinomas.

INVESTIGATION OF THE EFFECT OF SMALL THERMAL NEUTRON FLUXES ON THE PROPERTIES OF BACTERIA RHIZOBIUM PHASEOLI IS TAAS-80 TJ AND BACILLUS MEGATERIUM VAR. PHOSPHATICUM

In this work, the effects of a small thermal neutron flux on the growth rate of Rhizobium phaseoli IS TAAS-80 TJ and Bacillus megaterium var. phosphaticum bacteria intended for use in the production of micronutrients were studied. The phenomenon of germination of grow rate of bacteria under irradiation with small fluxes of thermal neutrons has been discovered. The best result was obtained with thermal neutron fluxes of 5.4-106 Fluence, at which the grow rate of bacteria increases by 5-6 times. It has been experimentally shown that one of the possible mechanisms of the influence of thermal neutrons on the properties of biological objects is the interaction of thermal neutrons by the nuclei of light atoms of the composition of these objects. For this purpose, the IR spectra of the bacteria Rhizobium phaseoli IS TAAS-80 TJ and Bacillus megaterium var. phosphaticum were studied and the effect of a small thermal neutron flux on the spectral characteristics of these bacteria was estimated. It was found that in some bands of the IR spectrum of bacteria, when they are irradiated with small fluxes of thermal neutrons, the optical density intensity ratio changes. The greatest change is observed with thermal neutron fluxes of 5.4-106 Fluence, which correlates with an increase in the growth rate of bacteria by 5-6 times. It has been established that the change in the biological activity of bacteria is associated with a change in methyl groups. It has been experimentally shown that one of the possible mechanisms of the influence of thermal neutrons on the IR spectrum of the bacteria Rhizobium phaseoli IS TAAS-80 TJ and Bacillus megaterium var. phosphaticum is a nuclear reaction of radiation capture of thermal neutrons by nuclei of nitrogen atoms of the species . It is assumed that the mechanism of changing the activity of bacteria under the influence of thermal neutrons is due to a change in the conformation of DNA chains at certain sites and methylation of DNA, proteins and polymerase.

Methylation of the epigenetic JMJD2D protein by SET7/9 promotes prostate tumorigenesis.

How the function of the JMJD2D epigenetic regulator is regulated or whether it plays a role in prostate cancer has remained elusive. We found that JMJD2D was overexpressed in prostate tumors, stimulated prostate cancer cell growth and became methylated by SET7/9 on K427. Mutation of this lysine residue in JMJD2D reduced the ability of DU145 prostate cancer cells to grow, invade and form tumors and elicited extensive transcriptomic changes. This included downregulation of CBLC, a ubiquitin ligase gene with hitherto unknown functions in prostate cancer, and upregulation of PLAGL1, a transcription factor with reported tumor suppressive characteristics in the prostate. Bioinformatic analyses indicated that CBLC expression was elevated in prostate tumors. Further, downregulation of CBLC largely phenocopied the effects of the K427 mutation on DU145 cells. In sum, these data have unveiled a novel mode of regulation of JMJD2D through lysine methylation, illustrated how this can affect oncogenic properties by influencing expression of the CBLC gene, and established a pro-tumorigenic role for CBLC in the prostate. A corollary is that JMJD2D and CBLC inhibitors could have therapeutic benefits in the treatment of prostate and possibly other cancers.

Polymorphism and promoter methylation-regulated mRNA expression of IFI6 gene affect meat quality in pigs

This study examined the association, promoter methylation of Interferon alpha-inducible protein 6 (IFI6) gene, and their effects on mRNA expression and meat quality traits in pig. Muscle samples (n=300) were randomly collected from the Longissimus thoracis et lumborum (LTL) to determine meat quality traits including drip loss, thawing loss, cooking loss, meat pH [at 45 minutes (pH45min) and at 24 hours (pH24h) post-mortem], shear force (for tenderness), and lightness (L*) in pigs. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Muscle samples (5 per group) with divergent meat pH (low vs high) were selected for promoter methylation study using bisulfate and pyrosequencing. Muscle samples from animals (5 per group) with divergent phenotypes (low vs high meat pH and low vs high drip loss) were used for mRNA expression study by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that a polymorphism in IFI6 gene (g.370A>G) was significantly (P < 0.01) associated with meat pH24h. Furthermore, four CpG regions were identified in IFI6 promoter, of which DNA methylation in a CG site was higher in low meat pH24h group compared to high meat pH24h group (P < 0.05). The mRNA expression of IFI6 gene was significantly (P < 0.01) downregulated in low meat pH24h group than high meat pH24h group. This result postulated that high DNA methylation at CG site in the promoter region might downregulate the IFI6 gene expression, hence lower the muscle pH that might lead to higher drip loss and lower tenderness. Therefore, porcine IFI6 gene might be a potential candidate gene for meat quality trait in pig. Nevertheless, further research is needed to confirm the role of IFI6 gene in controlling meat quality traits in other commercial populations.

Comprehensive Analysis of CircRNA Expression Profiles in Multiple Tissues of Pigs.

Circular RNAs (circRNAs) are a class of non-coding RNAs with diverse functions, and previous studies have reported that circRNAs are involved in the growth and development of pigs. However, studies about porcine circRNAs over the past few years have focused on a limited number of tissues. Based on 215 publicly available RNA sequencing (RNA-seq) samples, we conducted a comprehensive analysis of circRNAs in nine pig tissues, namely, the gallbladder, heart, liver, longissimus dorsi, lung, ovary, pituitary, skeletal muscle, and spleen. Here, we identified a total of 82,528 circRNAs and discovered 3818 novel circRNAs that were not reported in the CircAtlas database. Moreover, we obtained 492 housekeeping circRNAs and 3489 tissue-specific circRNAs. The housekeeping circRNAs were enriched in signaling pathways regulating basic biological tissue activities, such as chromatin remodeling, nuclear-transcribed mRNA catabolic process, and protein methylation. The tissue-specific circRNAs were enriched in signaling pathways related to tissue-specific functions, such as muscle system process in skeletal muscle, cilium organization in pituitary, and cortical cytoskeleton in ovary. Through weighted gene co-expression network analysis, we identified 14 modules comprising 1377 hub circRNAs. Additionally, we explored circRNA-miRNA-mRNA networks to elucidate the interaction relationships between tissue-specific circRNAs and tissue-specific genes. Furthermore, our conservation analysis revealed that 19.29% of circRNAs in pigs shared homologous positions with their counterparts in humans. In summary, this extensive profiling of housekeeping, tissue-specific, and co-expressed circRNAs provides valuable insights into understanding the molecular mechanisms of pig transcriptional expression, ultimately deepening our understanding of genetic and biological processes.

Role of methyltransferase Set7 in obesity-related endothelial activation: functions beyond histone methylation

Abstract Introduction Obesity is associated with environmental changes such as hyperglycemia, hyperinsulinemia, inflammation, and elevated free fatty acids, all of which compromise endothelial function1,2. We have previously reported that in individuals with type 2 diabetes, lysine methyltransferase Set7 is involved in hyperglycemia-induced endothelial damage through monomethylation of histone 3 at lysine 4 (H3K4me1)3. Apart from such histone-driven epigenetic modulation, Set7-induced methylation of non-histone proteins is emerging as a novel mechanism underlying the (dys)regulation of many biological processes4. Purpose To investigate the role of Set7-related endothelial damage in cells exposed to different obesity-associated stimuli and to determine its impact on the protein methylome in these settings. Methods Human aortic endothelial cells (HAECs) were exposed to high concentrations of glucose, insulin, TNFa, or fatty acids (palmitate or oleate). Set7 overexpression, silencing, and inhibition studies were also performed in these cells. Gene and protein expression were assessed by RT-PCR and immunoblotting respectively, whereas production of the reactive oxygen species (ROS) was measured by electron spin resonance spectroscopy. Specific antibodies were used for the enrichment of lysine mono-, di-, and tri-methylated proteins and LC-MS/MS proteomic analysis was carried out for their identification. Results Each obesity model in HAECs increased the expression of nearly all the inflammatory markers tested and, except for TNFa, they also increased the expression of Set7. Significant changes in the global levels of H3K4me1, as well as of methylated proteins were also observed in these cells. Studies overexpressing a mutant form of Set7, which is lacking the SET domain, revealed that Set7 methyltransferase activity is required to define the protein methylome in HAECs. Similar results were obtained after Set7 silencing and inhibition. Despite no differences were observed in the production of ROS when modulating Set7 expression in HAECs, overexpression of an active form of Set7 and its silencing/inhibition resulted in a consistent up- and down-regulation, respectively, of several inflammatory mediators and adhesion molecules. Finally, proteomics analysis identified, for the first time, the protein methylome of endothelial cells, comprising almost 2,700 methylated proteins involved in essential biological processes such as RNA metabolism and cytoskeleton. Conclusions Our results indicate that Set7 may contribute to inflammatory changes in response to obesogenic insults. Moreover, our findings uncover a dual role of methyltransferase Set7 in governing the methylation of both histone and non-histone proteins in endothelial cells. These preliminary findings open a new field of research aiming to further elucidate the molecular mechanisms underlying the modulation of the protein methylome in the endothelium and its role in endothelial damage.

Cuproptosis-related gene DLAT serves as a prognostic biomarker for immunotherapy in clear cell renal cell carcinoma: multi-database and experimental verification.

Renal clear cell carcinoma (ccRCC) is the most common type of renal cancer. Here we aim to explore the prognosis and immunotherapeutic value of copper death-related gene Dihydrolipoamide S-acetyltransferase (DLAT) in ccRCC. The mRNA and protein expressions and methylation level of DLAT, as well as the relation of DLAT to survival prognosis, clinical characteristics, biological function, and immune microenvironment and responses in patients with ccRCC were evaluated using multiple databases. In addition, 75 paired ccRCC tissue samples and 3 kinds of cell lines were tested for experimental validation. Bioinformatics analysis of multiple databases, qRT-PCR, and western blot verified that DLAT expression in ccRCC was lower than that in paracancerous tissues. Patients with low expression of DLAT had a lower survival rate, worse clinical prognosis, stronger immune cell infiltration and expression of immunosuppressive points, and higher tumor immune dysfunction and exclusion (TIDE) scores. DLAT was identified as an independent prognostic factor in ccRCC and was closely related to the prognosis and immune responses of patients with ccRCC.

72 Effects of Differing Planes of Nutrition and one-Carbon Metabolite Supplementation of Beef Heifers on B12, Folate, and Folate Intermediate Concentrations in the dam and Fetus

Abstract Vitamin B12 and folate are involved in the metabolic process known as one-carbon metabolism, which among other things, has a role in DNA, RNA, and protein methylation. This methylation connects one-carbon metabolism to epigenetics, which influences fetal development. Thus, an adequate supply of one-carbon metabolites (OCM) is important during fetal development. We hypothesized that differing planes of nutrition, along with supplementation of OCM, would impact concentrations of B12, folate, and folate intermediates in maternal liver, maternal serum, fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF). Specifically, OCM supplementation would increase circulating and hepatic B12 and folate concentrations compared with non-supplemented heifers. Heifers (n = 72) were individually fed on one of two planes of nutrition: control (CON; 0.45 kg/d gain) or restricted (RES; -0.23 kg/d gain). To complete the 2 × 2 factorial arrangement, heifers were also given either OCM supplementation (10 g/d rumen-protected methionine + 60 g/d rumen-protected choline in the diet plus 20 mg B12 + 320 mg folic acid; weekly intramuscular injections) or no supplementation (grain carrier + saline injection). Thus, the final treatments were CON-OCM, CON+OCM, RES-OCM, and RES+OCM. Heifers were bred to a single sire via artificial insemination with sexed semen. Heifers pregnant with female calves (n = 31) were selected for slaughter on d 63. Samples of maternal liver, ALF, AMF, and fetal liver were collected at slaughter. Maternal serum was collected at breeding (d 0) and at slaughter. For B12 and folate concentration analysis, maternal serum was sent to IDEXX BioAnalytics (N. Grafton, MA). For analysis of folate intermediate concentrations in maternal and fetal liver, ALF, and AMF, samples were analyzed using LC-MS/MS. There were treatment × day interactions (P ≤ 0.05) for B12 and folate in maternal serum; on d 63, R+OCM was greater compared with C-OCM and R-OCM and all treatments at d 0 (Table 1). For folate intermediate concentrations, no plane of nutrition × supplement interactions (P ≥ 0.06) were found in fetal liver, ALF, or AMF; however, 5-methyl THF in ALF tended (P = 0.06) to be greater in R+OCM compared with all other treatments (Table 2). No interactions were found for concentrations in maternal liver when corrected for total liver weight; however, there was a supplement effect (P ≤ 0.05) where the concentrations of folic acid, dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate were increased in +OCM compared with -OCM, and a plane of nutrition effect (P ≤ 0.05) with increased 5,10-methenyltetrahydrofolate and 5,10-methylenetetrahydrofolate concentrations in RES. We conclude that supplementing OCM increases concentrations of B12, folate and its intermediates in maternal liver and serum. USDA is an equal opportunity provider and employer.

Resistance to 2-Hydroxy-Flutamide in Prostate Cancer Cells Is Associated with the Downregulation of Phosphatidylcholine Biosynthesis and Epigenetic Modifications.

In this study, we examined the metabolic adaptations of a chemoresistant prostate cancer cell line in comparison to a sensitive cell line. We utilized prostate cancer LNCaP cells and subjected them to a stepwise increase in the antiandrogen 2-hydroxy-flutamide (FLU) concentration to generate a FLU-resistant cell line (LN-FLU). These LN-FLU cells displayed characteristics of cancer stem cells, exhibited drug resistance, and showed a significantly reduced expression of Cyclin D1, along with the overexpression of p16, pointing to a proliferation arrest. In comparing the cancer stem-like LN-FLU cells to the LNCaP cells, we observed a decrease in the expression of CTP-choline cytidylyl transferase α (CCTα), as well as a decline in choline kinase, suggesting altogether a downregulation of the phosphatidylcholine biosynthetic pathway. In addition, we found decreased levels of the protein methyl transferase PRMT2 and the upregulation of the histone deacetylase Sirtuin1 (Sirt1). Analysis of the human prostate cancer samples revealed similar results in a population with high expressions of the stem cell markers Oct4 and ABCB1A1. Our findings suggest that the adaptation of prostate cancer cells to antiandrogens could induce reprogramming into stem cells that survive in a low phosphocholine metabolism and cell cycle arrest and display drug resistance.

PRMT5 mediates FoxO1 methylation and subcellular localization to regulate lipophagy in myogenic progenitors

Development is regulated by various factors, including protein methylation status. While PRMT5 is well known for its roles in oncogenesis by mediating symmetric di-methylation of arginine, its role in normal development remains elusive. Using Myod1Cre to drive Prmt5 knockout in embryonic myoblasts (Prmt5MKO), we dissected the role of PRMT5 in myogenesis. The Prmt5MKO mice are born normally but exhibit progressive muscle atrophy and premature death. Prmt5MKO inhibits proliferation and promotes premature differentiation of embryonic myoblasts, reducing the number and regenerative function of satellite cells in postnatal mice. Mechanistically, PRMT5 methylates and destabilizes FoxO1. Prmt5MKO increases the total FoxO1 level and promotes its cytoplasmic accumulation, leading to activation of autophagy and depletion of lipid droplets (LDs). Systemic inhibition of autophagy in Prmt5MKO mice restores LDs in myoblasts and moderately improves muscle regeneration. Together, PRMT5 is essential for muscle development and regeneration at least partially through mediating FoxO1 methylation and LD turnover.

Adenosine dialdehyde, a methyltransferase inhibitor, induces colorectal cancer cells apoptosis by regulating PIMT:p53 interaction

Post-translational modification (PTM) is important in controlling many biological processes by changing the structure and function of a protein. Protein methylation is an important PTM, and the role of methyltransferases has been implicated in numerous cellular functions. Protein L-isoaspartyl methyltransferase (PIMT) is ubiquitously expressed in almost all organisms and govern important cellular processes including apoptosis. Among other functions, PIMT has also been identified as a potent oncogene because it destabilizes the structure of the tumor suppressor p53 via methylation at the transactivation domain. In the present study we identified that out of the three methyltransferase inhibitors tested, namely, S-adenosyl-l-homocysteine (AdoHcy), adenosine and adenosine dialdehyde (AdOx), only AdOx augments p53 expression by destabilizing PIMT structure, as evident from far-UV CD. The effect of the inhibitors, AdOx in particular, to the structure of PIMT, and the binding of PIMT to the p53 transactivation domain have been investigated by docking and molecular dynamics simulations. AdOx significantly increases p53 accumulation and nuclear translocation in colon cancer cells, triggering the p53-mediated apoptotic pathway. To better understand the molecular mechanisms underlying p53 accumulation in colon cancer cells, we observed that the level of PIMT is considerably lower in AdOx-treated cells, reducing its association with p53, which stabilized p53. p53 then transactivated BAX, increasing the BAX: BCL-2 ratio and causing colon cancer cell death.

Protein methylation characterization using NMR without isotopic labeling

Protein methylation is crucial in epigenetics, and targeting the involved methyltransferases shows great potential for therapeutic intervention with several inhibitors in clinical trials for oncology indications. Therefore, characterization of protein methylation is essential for understanding the methyltransferase function and discovering chemical inhibitors and antagonists. While NMR has been used to measure methylation rates, isotopic labeling of protein or methyl donors can be costly and cannot characterize demethylation of proteins extracted from natural sources. Our method employs a four-quantum filter 1H-13C experiment that selectively detects methyl groups, providing a simple way to characterize methylation and demethylation features of methyltransferases and demethylases, respectively, without requiring isotopic labeling. In our experiments, we successfully observed the methylation of H3 under lysate from various cells and tissues of mice with cancerous growth. The results revealed that H3 undergoes both mono- and dimethylation in all the tested lysates, but at varying rates and degrees. Significantly lower H3 methylation rates and levels were observed in both cervical tumor and breast tumor lysates compared with the corresponding cancerous cells and healthy cells lysates. These findings highlight the variability of histone H3 methylation patterns among healthy cells, cancerous cells, tumor tissues, and different tumor types, and suggest that this method has great potential in facilitating the development of effective interventions against these diseases. By characterizing the methylation features of suspected tumors or areas of concern, it provides valuable insights into the underlying mechanisms of cancer development and aids in identifying potential targets for therapeutic interventions.

PRMT6 methylation of STAT3 regulates tumor metastasis in breast cancer

Overcoming distant metastasis stands as a paramount challenge in enhancing the outcomes of breast cancer treatments. Thus, delving deeper into comprehending the intricate mechanisms underlying breast cancer metastasis becomes imperative, offering potential avenues for pioneering therapeutic approaches. PRMT6, an arginine N-methyltransferase, possesses the ability to methylate both histone and non-histone proteins. It has been reported that methylation of non-histone proteins impacts their cellular localization, stability, and activation, consequently influencing tumor progression. However, the extent to which PRMT6-mediated non-histone protein methylation influences cancer cell metastasis, particularly in the context of breast cancer, remains elusive. In this study, we established that PRMT6 exerted a positive regulatory influence on breast cancer metastasis through both in vivo and in vitro experiments. Mechanistically, we innovatively revealed that PRMT6 asymmetrically di-methylated STAT3 at arginine 729 (STAT3 R729me2a). This modification proved indispensable for STAT3’s membrane localization, its interaction with JAK2, STAT3 Y705 phosphorylation, and PRMT6-driven cancer cell metastasis. From a clinical perspective, we unearthed the promising potential of STAT3 R729me2a as a robust prognostic marker for predicting the overall survival time of breast cancer patients. In terms of therapeutic intervention, we demonstrated the significant capability of the PRMT6 inhibitor, EPZ020411, to curtail breast cancer metastasis both in vivo and in vitro. In sum, our study unveils the pivotal biological role of PRMT6-mediated STAT3 R729me2a in breast cancer metastasis and underscores the prospective utility of PRMT6 inhibitors as effective therapeutic strategies against STAT3-driven metastatic breast cancer.