Epigenetic Regulation Of Gene Expression Research Articles

Protein arginine methylation in transcription and epigenetic regulation

Arginine methylation is a prevalent post-translational modification found in all eukaryotic systems. It involves the addition of a methyl group to the guanidino nitrogen atoms of arginine residues within proteins, and this process is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). In mammals, there exist nine PRMTs (PRMT1–9) that catalyze three distinct types of arginine methylation: monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine. These modifications play critical roles in numerous fundamental cellular processes, including transcription, RNA metabolism, genome maintenance, and signaling transduction. Aberrations in protein arginine methylation have been implicated in various human diseases, such as neurodevelopmental disorders and cancer. This review offers a general overview of arginine methylation, covering its deposition, its impact on protein function, and the diverse regulatory mechanisms involved. We specifically focus on an in-depth view of the role of arginine methylation in transcription and the epigenetic regulation of gene expression. Readers are directed towards additional reviews that encompass other aspects of arginine methylation biology.

Micronutrients in High-Fat Diet Modify Insulin Resistance and Its Regulatory Genes in Adult Male Mice.

Obesity and insulin resistance (IR) are associated with epigenetic changes of gene expression. However, the relationship between micronutrients, epigenetic regulation of gene expression, and IR during development of diet-induced obesity has yet to be defined. Our objective is to describe the effect of micronutrient addition to diets on IR and its related genes during obesity development. Male C57BL/6J mice are fed a high-fat (HFD) or low-fat (LFD) diets with or without a multi-vitamin mineral mix (MVM) addition containing vitamins A, B1, B6, B12, and Zn, and Se for 9 weeks. Compared to LFD mice, HFD mice have higher body weight, IR, fasting glucose, insulin, C-peptide, leptin, and hepatic triglyceride concentrations, and dysregulated gene expression in liver, muscle, pancreas, and fat tissues (p < 0.05). The addition of MVM reduces these HFD-induced effects. HFD downregulates 27 genes associated with insulin regulation and adipose tissue function across all tissues by an average of 47% and upregulates five genes by 230% (p < 0.001). Adding MVM downregulates five genes and upregulates one in HFD-fed mice. Both HFD and MVM alter one-carbon metabolites. Addition of micronutrients to the HFD decreases IR and modifies associated gene expression in obese and lean mice.

Interaction of DNA Methyltransferase Dnmt3a with Phosphorus Analogs of S-Adenosylmethionine and S-Adenosylhomocysteine

Enzymatic methyltransferase reactions are of crucial importance for cell metabolism. S-Adenosyl-L-methionine (AdoMet) is a main donor of the methyl group. DNA, RNA, proteins, and low-molecular-weight compounds are substrates of methyltransferases. In mammals, DNA methyltransferase Dnmt3a de novo methylates the C5 position of cytosine residues in CpG sequences in DNA. The methylation pattern is one of the factors that determine the epigenetic regulation of gene expression. Here, interactions with the catalytic domain of Dnmt3a was for the first time studied for phosphonous and phosphonic analogs of AdoMet and S-adenosyl-L-homocysteine (AdoHcy), in which the carboxyl group was substituted for respective phosphorus-containing group. These AdoMet analogs were shown to be substrates of Dnmt3a, and the methylation efficiency was only halved as compared with that of natural AdoMet. Both phosphorus-containing analogs of AdoHcy, which is a natural methyltransferase inhibitor, showed similar inhibitory activities toward Dnmt3a and were approximately four times less active than AdoHcy. The finding that the phosphonous and phosphonic analogs are similar in activity was quite unexpected because the geometry and charge of their phosphorus-containing groups differ substantially. The phosphorus-containing analogs of AdoMet and AdoHcy are discussed as promising tools for investigation of methyltransferases.

MicroRNA as epigenetic regulators of canine cryptorchidism

Cryptorchidism, the failed descent of one or both testes into the scrotum, is a common developmental disorder in male dogs. Cryptorchidism may affect canine fertility, reducing the quality of the semen, and may promote spermatic cord torsion and onset of neoplasia. MicroRNAs (miRNAs) are epigenetic regulators of gene expression and their dysregulation is associated with disorders of spermatogenesis and testis neoplasia. The present study aimed at investigating the expression of miRNAs in formalin-fixed, paraffin-embedded (FFPE) canine retained testes and testes affected by seminoma, and at integrating miRNAs to their target genes. Forty testicular FFPE specimens from 30 dogs were included - 10 scrotal and 10 contralateral retained from 10 unilateral cryptorchid dogs; 10 tumoral testes affected by seminoma from non-cryptorchid dogs; 10 scrotal normal testes from non-cryptorchid dogs included as the control. The expression level of three miRNAs, namely miR-302c-3p, miR-302a-3p, and miR-371-3p, associated with testicular disorders, were quantified using RT-qPCR.The comparative analysis demonstrated that the level of miR-302a-3p and miR-371a-3p were quantifiable exclusively in control testes. The expression level of miR-302c-3p was higher in the control than in the other groups; its expression decreased in retained testes compared to scrotal testes and testes with seminoma. Gene Ontology analysis pointed out that these miRNAs may be involved in the modulation of estrogen and thyroid hormone signaling pathways.In conclusion, this study demonstrated that miRNAs are dysregulated in canine cryptorchid and seminoma-affected testes compared to control tissues, confirming the pivotal role of miRNAs in cryptorchidism.

HDAC inhibition regulates oxidative stress in CD4+Thelper cells of chronic obstructive pulmonary disease and non-small cell lung cancer patients via mitochondrial transcription factor a (mtTFA) modulating NF-κB/HIF1α axis

Histone deacetylases (HDACs) play a crucial role in the epigenetic regulation of gene expression by remodelling chromatin. Isoenzymes of the HDAC family exhibit aberrant regulation in a wide variety of cancers as well as several inflammatory lung disorders like chronic obstructive pulmonary disease (COPD). Inhibition of HDACs is a potential therapeutic strategy that could be used to reverse epigenetic modification. Trichostatin A (TSA), a powerful histone deacetylase (HDAC) inhibitor, has anti-cancer effects in numerous cancer types. However, it is not yet apparent how HDAC inhibitors affect human non-small cell lung cancer cells (NSCLC) and COPD. This study aims to investigate TSA's role in restoring mitochondrial dysfunction and its effect on hypoxia and inflammation in CD4+T cells obtained from patients with COPD and lung cancer. As a result of treatment with TSA, there is a reduction in the expression of inflammatory cytokines and a decreased enrichment of transcriptional factors associated with inflammation at VEGFA gene loci. We have seen a substantial decrease in the expression of NF-κB and HIF1α, which are the critical mediators of inflammation and hypoxia, respectively. Following TSA treatment, mtTFA expression was increased, facilitating patients with COPD and NSCLC in the recovery of their dysfunctional mitochondria. Furthermore, we have discovered that TSA treatment in patients with COPD and NSCLC may lead to immunoprotective ness by inducing Th1ness. Our finding gives a new insight into the existing body of knowledge regarding TSA-based therapeutic methods and highlights the necessity of epigenetic therapy for these devastating lung disorders.

Genome-Wide Acetylation Modification of H3K27ac in Bovine Rumen Cell Following Butyrate Exposure.

Butyrate contributes epigenetically to the changes in cellular function and tissue development of the rumen in ruminant animals, which might be achieved by its genetic or epigenetic regulation of gene expression. To explore the role of butyrate on bovine rumen epithelial function and development, this study characterized genome-wide H3K27ac modification changes and super-enhancer profiles in rumen epithelial primary cells (REPC) induced with butyrate by ChIP-seq, and analyzed its effects on gene expression and functional pathways by integrating RNA-seq data. The results showed that genome-wide acetylation modification was observed in the REPC with 94,675 and 48,688 peaks in the butyrate treatment and control group, respectively. A total of 9750 and 5020 genes with increased modification (H3K27ac-gain) and decreased modification (H3K27ac-loss) were detected in the treatment group. The super-enhancer associated genes in the butyrate-induction group were involved in the AMPK signaling pathway, MAPK signaling pathway, and ECM-receptor interaction. Finally, the up-regulated genes (PLCG1, CLEC3B, IGSF23, OTOP3, ADTRP) with H3K27ac gain modification by butyrate were involved in cholesterol metabolism, lysosome, cell adhesion molecules, and the PI3K-Akt signaling pathway. Butyrate treatment has the role of genome-wide H3K27ac acetylation on bovine REPC, and affects the changes in gene expression. The effect of butyrate on gene expression correlates with the acetylation of the H3K27ac level. Identifying genome-wide acetylation modifications and expressed genes of butyrate in bovine REPC cells will expand the understanding of the biological role of butyrate and its acetylation.

Identification of methylation pattern in the partial promoter of acetyl CoA carboxylase beta (ACACB) gene in White Leghorn line

The present study was initiated with an objective of identifying methylation pattern in the partial promoter of Acetyl CoA carboxylase beta (ACACB) gene in White Leghorn IWK line. To understand the epigenetic regulation of gene expression, birds with highest and lowest expression at 18th week and 40th week age were chosen. Epigenetic profile of the minimal promoter indicated that there were seven CpG islands in this region, positioned at 75, 87, 155, 269, 284, 311 and 426 positions of the 555 bp promoter region. Among these, except the position 75, the remaining positions showed cis-acting transcription factors. There was negative relationship between highest and lowest expressed birds with the per cent methylation in White Leghorn IWK line. The mean methylation per cent was found to be 42.85 and 21.42 at 18th and 40th weeks of age, respectively. The results indicated decrease in methylation per cent with age. The overall mean methylation per cent of 32.14 was observed.

MicroRNA expression associated with low-grade cervical intraepithelial neoplasia outcomes.

Only a fraction of low-grade cervical intraepithelial neoplasia (CIN) progresses to high-grade CIN; however, the biological processes that differentiate progressive CIN from CIN that resolves naturally are poorly understood. MicroRNAs (miRNAs) are important epigenetic regulators of gene expression and thus, miRNA expression profiling can reveal the dysregulated biology underlying disease processes. The purpose of this case-control study was to reveal miRNA expression patterns and predict the underlying biological pathways that are associated with clinical outcomes of low-grade CIN. Women with low-grade CIN diagnosis and definitive clinical outcomes (n = 51) were identified retrospectively using electronic clinical records. Comprehensive miRNA expression profiling was performed on the low-grade CIN diagnostic cervical biopsies retrieved from pathology archives. Differential miRNA expression was analyzed by comparing women with CIN that progressed to women with CIN that resolved naturally. Differential expression of 29 miRNAs was observed in low-grade CIN that progressed to high-grade compared to low-grade CIN that resolved. Of these, 24 were significantly downregulated in progressive CIN, including miR-638, miR-3196, miR-4488, and miR-4508, while 5 miRNAs, including miR-1206a, were significantly upregulated. Computational gene ontology analysis based on the discovered miRNAs and their putative mRNA targets revealed biological processes associated with oncogenic phenotypes. Distinct miRNA expression profiles are associated with clinical outcomes of low-grade CIN. The functional effects of the differentially expressed miRNAs may be biological determinants of CIN progression or resolution.

Integrative profiling of extrachromosomal circular DNA in placenta and maternal plasma provides insights into the biology of fetal growth restriction and reveals potential biomarkers.

Introduction: Fetal growth restriction (FGR) is a placenta-mediated pregnancy complication that predisposes fetuses to perinatal complications. Maternal plasma cell-free DNA harbors DNA originating from placental trophoblasts, which is promising for the prenatal diagnosis and prediction of pregnancy complications. Extrachromosomal circular DNA (eccDNA) is emerging as an ideal biomarker and target for several diseases. Methods: We utilized eccDNA sequencing and bioinformatic pipeline to investigate the characteristics and associations of eccDNA in placenta and maternal plasma, the role of placental eccDNA in the pathogenesis of FGR, and potential plasma eccDNA biomarkers of FGR. Results: Using our bioinformatics pipelines, we identified multi-chromosomal-fragment and single-fragment eccDNA in placenta, but almost exclusively single-fragment eccDNA in maternal plasma. Relative to that in plasma, eccDNA in placenta was larger and substantially more abundant in exons, untranslated regions, promoters, repetitive elements [short interspersed nuclear elements (SINEs)/Alu, SINEs/mammalian-wide interspersed repeats, long terminal repeats/endogenous retrovirus-like elements, and single recognition particle RNA], and transcription factor binding motifs. Placental multi-chromosomal-fragment eccDNA was enriched in confident enhancer regions predicted to pertain to genes in apoptosis, energy, cell growth, and autophagy pathways. Placental eccDNA-associated genes whose abundance differed between the FGR and control groups were associated with immunity-related gene ontology (GO) terms. The combined analysis of plasma and placental eccDNA-associated genes in the FGR and control groups led to the identification of potential biomarkers that were assigned to the GO terms of the epigenetic regulation of gene expression and nutrient-related processes, respectively. Conclusion: Together, our results highlight links between placenta functions and multi-chromosomal-fragment and single-fragment eccDNA. The integrative analysis of placental and plasma eccDNA confirmed the potential of these molecules as disease-specific biomarkers of FGR.

Involvement of the H3.3 Histone Variant in the Epigenetic Regulation of Gene Expression in the Nervous System, in Both Physiological and Pathological Conditions.

All the cells of an organism contain the same genome. However, each cell expresses only a minor fraction of its potential and, in particular, the genes encoding the proteins necessary for basal metabolism and the proteins responsible for its specific phenotype. The ability to use only the right and necessary genes involved in specific functions depends on the structural organization of the nuclear chromatin, which in turn depends on the epigenetic history of each cell, which is stored in the form of a collection of DNA and protein modifications. Among these modifications, DNA methylation and many kinds of post-translational modifications of histones play a key role in organizing the complex indexing of usable genes. In addition, non-canonical histone proteins (also known as histone variants), the synthesis of which is not directly linked with DNA replication, are used to mark specific regions of the genome. Here, we will discuss the role of the H3.3 histone variant, with particular attention to its loading into chromatin in the mammalian nervous system, both in physiological and pathological conditions. Indeed, chromatin modifications that mark cell memory seem to be of special importance for the cells involved in the complex processes of learning and memory.

P-219 Single-follicle spatial proteomics of the whole ovary by MALDI mass spectrometry imaging: towards the identification of protein-markers of the follicle's quality

Abstract Study question Can we improve our knowledge of the single-follicle proteome signature by MALDI mass spectrometry imaging? Summary answer We identified follicle-type proteins that function during follicle growth (NUMA1, TPM2), GV-to-MII transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2) and preimplantation development (TIF1B, KHDC3). What is known already The acquisition of oocytes developmental competence occurs through a bidirectional exchange of molecular information between the oocyte, the companion follicle cells, the stroma, and the vascular network. This reciprocal relationship is still poorly understood and particularly scarce is our knowledge of the proteins that are playing a regulative role during folliculogenesis. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool that allows the study of the proteomic landscape of small morphological structures inside a tissue, but in the field of reproduction has been only used to analyse rodent spermatogenesis. Study design, size, duration Nano-scale liquid chromatography-electrospray ionisation-tandem mass spectrometry (nLC-ESI-MS/MS) was combined with MALDI-MSI to identify the proteomic landscape and to map the changes occurring throughout folliculogenesis. We performed single-follicle spatial proteomics on all the follicles, from the secondary to the fully-grown preovulatory, present in histological serial sections of an entire prepubertal 25-day-old mouse ovary. Cross-referencing our results with those obtained by previous proteomics and transcriptomics analyses allowed to strengthen their significance. Participants/materials, setting, methods A single 25-day-old mouse ovary was fixed in 10% formalin, dehydrated, embedded in paraffin, and sectioned to obtain 133 6-μm serial sections. After paraffin removal, trypsin digestion and matrix deposition, mass spectra were acquired using a rapifleX MALDI Tissuetyper™ (Bruker) with a raster sampling of 20 μm in both x and y axes. Then, tryptic peptides were extracted from sections and analysed with nLC-ESI-MS/MS using a Dionex-UltiMate-3000 LC-nano-system coupled with an Impact HD™ UHR-QqToF (Bruker). Main results and the role of chance This study proposes a spatial proteomics workflow to investigate the proteome of a whole prepubertal 25-day-old mouse ovary, preserving the spatial relationship of the peptides in this complex histological context. A total of 401 proteins were identified by nLC-ESI-MS/MS, 69 with a known function in ovary biology. Enrichment analysis highlighted significant KEGG and Reactome pathways, with apoptosis, developmental biology, PI3K-Akt, epigenetic regulation of gene expression, and extracellular matrix organisation being well represented. Then, correlating these data with the spatial information provided by MALDI-MSI on 276 follicles highlighted 94 proteins that were detected throughout the secondary to the pre-ovulatory transition. Of these, 37 proteins showed a gradual quantitative change during follicle differentiation, comprising 10 with a known role in follicle growth (NUMA1, TPM2), oocyte GV-to-MII transition (SFPQ, ACTBL, MARCS, NUCL), ovulation (GELS, CO1A2) and preimplantation development (TIF1B, KHDC3). The proteome landscape identified includes molecules of known function in the ovary, but also those whose specific role is emerging. Altogether, this work demonstrates the utility of performing spatial proteomics in the context of the ovary and offers sound bases for more in-depth investigations that aim to further unravel its spatial proteome. Limitations, reasons for caution A limitation of MALDI-MSI is its 20 µm/pixel resolution which 1) leads to the acquisition of spectra comprising follicular and extra-follicular tissue, particularly with small follicles (15-30µm in diameter); and 2) does not distinguish among the different follicular cell types, thus precluding its application for single-cell in situ proteomics. Wider implications of the findings MALDI-MSI is a potent technology to study the proteome of ovary specimens, but it could also be used for the analysis of their lipidic or metabolic profiles. Our pipelline aim to identify protein-markers of the follicle's quality and, in turn, of the oocyte's developmental competence. Trial registration number not appplicable

The role of DNA methylation on gene expression in the vertebrae of ancestrally benzo[a]pyrene exposed F1 and F3 male medaka

ABSTRACT Benzo[a]pyrene (BaP) is ubiquitously present in the aquatic environment and has been identified as a bone toxicant. Previous studies have demonstrated that ancestral BaP exposure can cause transgenerational bone deformities in fish. Transgenerational effects are thought to be caused by heritable epigenetic changes, such as DNA methylation, histone modification, and non-coding RNAs. To investigate the role of DNA methylation in BaP-induced transgenerational skeletal deformities and the related transcriptomic changes in deformed vertebrae, we examined the vertebrae of male F1 and F3 medaka fish using high-throughput RNA sequencing (RNA-seq) and whole-genome bisulphite sequencing (WGBS). The histological results revealed that osteoblast numbers at the vertebral bone decreased in the BaP-derived F1 and F3 adult males in comparison with the control group. Differentially methylated genes (DMGs) associated with osteoblastogenesis (F1 and F3), chondrogenesis (F1 and F3), and osteoclastogenesis (F3) were identified. However, RNA-seq data did not support the role of DNA methylation in the regulation of genes involved in skeletogenesis since there was very little correlation between the level of differential methylation and gene expression profiles related to skeletogenesis. Although DNA methylation plays a major role in the epigenetic regulation of gene expression, the dysregulation of vertebral gene expression patterns observed in the current study is most likely to be mediated by histone modification and miRNAs. Notably, RNA-seq and WGBS data indicated that genes related to nervous system development are more sensitive to ancestral BaP exposure, indicating a more complex transgenerational phenotype in response to ancestral BaP exposure.

DIPG-39. SIGNALING NETWORKS IN PEDIATRIC-TYPE DIFFUSE HIGH GRADE GLIOMAS

Abstract Pediatric-type diffuse high grade gliomas are aggressive brain cancers in children which to date lack effective treatment options and remain largely understudied. Even though genomic markers including histone H3 K27M and G34R mutations have been identified, their functional implications on signaling networks remain to be described. Phosphoproteomics might be able to uncover activated signaling pathways which could open up new paths for treatment. Here we analyzed formalin-fixed paraffin-embedded (FFPE) sections from K27M mutated, G34R mutated and histone H3 wild type patient tumors (n = 14) using phosphoproteomics. Our workflow not only enables the comprehensive characterization of phosphoserines and threonines but also of phosphotyrosines which, despite being rare modifications (0.1-1% of all phosphorylations), are known to play an important role in cancer. We found that signaling networks were distinct between the different tumor subtypes. For phosphotyrosines, tumor subtype accounted for the largest source of variation in the data (44% of variance) as determined by principle component analysis. The separation by subtype could also be seen from serine and threonine phosphoproteomics and proteomics, but was not as striking as for the phosphotyrosine data. Differential abundance analysis, partial least squares discriminant analysis and self-organizing maps revealed deregulated signaling networks between subtypes. The most downregulated pathway in K27M compared to wild type tumors was enriched for proteins involved in the epigenetic regulation of gene expression (FDR&amp;lt;5%), which is in accordance with the remodeling of the epigenome taking place in K27M mutated tumors. In addition, we could identify signaling networks (e.g. EGFR signaling) which were activated in a few individual patients independent of their subtype and might be targetable. Our study provides insights into the signaling landscape of pediatric-type diffuse high grade gliomas. Recognizing deregulated signaling networks across subtypes and in individual patients could offer new avenues for personalized therapy.

Epigenomic analysis of Alzheimer's disease brains reveals diminished CTCF binding on genes involved in synaptic organization

Epigenetic aberrations are suggested to play an important role in transcriptional alterations in Alzheimer's disease (AD). One of the key mechanisms of epigenetic regulation of gene expression is through the dynamic organization of chromatin structure via the master genome architecture protein, CCCTC-binding factor (CTCF). By forming chromatin loops, CTCF can influence gene transcription in a complex manner. To find out whether genome-wide DNA binding sites for CTCF are altered in AD, we compared CTCF chromatin immunoprecipitation sequencing (ChIP-Seq) data from frontal cortex of human AD patients and normal controls (n = 9 pairs, all females). We have revealed that CTCF-binding affinity on many genes is significantly reduced in AD patients, and these genes are enriched in synaptic organization, cell adhesion, and actin cytoskeleton, including synaptic scaffolding molecules and receptors, such as SHANK2, HOMER1, NRXN1, CNTNAP2 and GRIN2A, and protocadherin (PCDH) and cadherin (CDH) family members. By comparing transcriptomic data from AD patients, we have discovered that many of the synaptic and adhesion genes with reduced CTCF binding in AD are significantly reduced in their mRNA expression. Moreover, a significant overlap of genes with the diminished CTCF binding and the reduced H3K27ac is identified in AD, with the common genes enriched in synaptic organization. These data suggest that the CTCF-controlled 3D chromatin organization is perturbed in AD, which may be linked to the diminished expression of target genes, probably through changes in histone modification.

How Does Nutrition Affect the Epigenetic Changes in Dairy Cows?

Dairy cows require a balanced diet that provides enough nutrients to support milk production, growth, and reproduction. Inadequate nutrition can lead to metabolic disorders, impaired fertility, and reduced milk yield. Recent studies have shown that nutrition can affect epigenetic modifications in dairy cows, which can impact gene expression and affect the cows' health and productivity. One of the most important epigenetic modifications in dairy cows is DNA methylation, which involves the addition of a methyl group to the DNA molecule. Studies have shown that the methylation status of certain genes in dairy cows can be influenced by dietary factors such as the level of methionine, lysine, choline, and folate in the diet. Other important epigenetic modifications in dairy cows are histone modification and microRNAs as regulators of gene expression. Overall, these findings suggest that nutrition can have a significant impact on the epigenetic regulation of gene expression in dairy cows. By optimizing the diet of dairy cows, it may be possible to improve their health and productivity by promoting beneficial epigenetic modifications. This paper reviews the main nutrients that can cause epigenetic changes in dairy cattle by analyzing the effect of diet on milk production and its composition.

Abstract 6018: Sequencing of 5-hydroxymethylcytosine to single base resolution

Abstract DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are identified using Illumina sequencing. Libraries are generated using either NEBNext® EM-seq™, an enzyme-based workflow, or by using sodium bisulfite conversion. These methods, however, cannot differentiate between 5mC and 5hmC. Distinguishing these modifications is important as there is increased interest in 5hmCs role in regulating gene expression. Methods currently exist to enable discrimination of 5mC and 5hmC, for example, oxBS-seq and TAB-seq. These methods still rely upon sodium bisulfite conversion and have reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC. 5hmC is detected using two enzymatic steps. Firstly, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. During Illumina sequencing deaminated cytosine and 5mC are read as thymine and the glucosylated 5hmC are read as cytosine. Subtractive analysis of 5hmC data from EM-seq data, which detects both 5mC and 5hmC, permits the identification of individual 5mC and 5hmC sites. 5hmC data were generated for inputs of 0.1 ng to 200 ng DNA isolated from E14 mouse embryonic stem cells and human brain. The 5hmC libraries had similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. T4147 phage DNA was used as an internal control, as all cytosines are 5-hydroxymethylated, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. The ability to discriminate between 5mC and 5hmC will provide key insights into the role of these cytosine modifications in development and disease. Citation Format: Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matthew A. Campbell, Nan Dai, Bradley W. Langhorst, Romualdas Vaisvila, Louise Williams. Sequencing of 5-hydroxymethylcytosine to single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6018.

Abstract 2235: A treatment for glioblastoma with a novel epigenetic inhibitor and identification of its target

Abstract Glioblastoma multiforme (GBM) is one of the deadliest human cancers. Despite extensive efforts to develop a new treatment, the prognoses of patients have not changed dramatically over the past several decades. LSD1 plays an essential role in the epigenetic regulation of gene expression in many cancer cells, including GBM, as it removes methyl groups from histone H3 lysine (K) 4me1/2 and H3K9me1/2. Since it was reported that suppression of LSD1 induced apoptosis of glioblastoma stem cells (GSCs), LSD1 has been expected as an effective therapeutic target for GBMs, particularly enriched with GSCs. We newly discovered an LSD inhibitor (S2172) that suppressed the LSD1 enzymatic activity effectively and sufficiently penetrated into brain. We found that S2172 showed a strong inhibitory effect on cell growth not only in GCS cells but also in differentiated GBM cells in vitro. In GBM cell line U251, IC50 of S2172 was lower than that of other LSD1 inhibitors (IC50 10.5 μM, 29.5 μM, 11.0 μM treated with S2172, Ory-1001, and CC-90011, respectively). We also found a robust suppression of transcription of oncogene Myc in differentiated GBM cells after S2172 treatment, which is highly expressed in most GBM cells and considered as a key element to maintain its stemness and proliferation. Consistent with the expression analysis, chromatin immunoprecipitation-sequencing (ChIP-seq) revealed that enrichment of H3K4m2 and H3K4m3 was efficiently altered by S2172 treatment along with the change of Myc expression. Finally, we treated U251 brain tumor mouse model with S2172. While S2172 had a strong effect in vitro, S2172 seemed unsuccessful in killing differentiated GBM cells in vivo. These data suggests that treatment of S2172 is less effective to differentiated GBM cells and this is consistent with previous report that LSD1 as a candidate therapeutic target in stem-like tumor propagating cells in GBM. Citation Format: Tokunaga Kota. A treatment for glioblastoma with a novel epigenetic inhibitor and identification of its target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2235.

Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

BackgroundT cell activation and programming from their naïve/resting state, characterized by widespread modifications in chromatin accessibility triggering extensive changes in transcriptional programs, is orchestrated by several cytokines and transcription regulators. PRDM1 and PRDM2 encode for proteins with PR/SET and zinc finger domains that control several biological processes, including cell differentiation, through epigenetic regulation of gene expression. Different transcripts leading to main protein isoforms with (PR +) or without (PR-) the PR/SET domain have been described. Although many studies have established the critical PRDM1 role in hematopoietic cell differentiation, maintenance and/or function, the single transcript contribution has not been investigated before. Otherwise, very few evidence is currently available on PRDM2. Here, we aimed to analyze the role of PRDM1 and PRDM2 different transcripts as mediators of T lymphocyte activation.MethodsWe analyzed the transcription signature of the main variants from PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) genes, in human T lymphocytes and Jurkat cells overexpressing PRDM2 cDNAs following activation through different signals.ResultsT lymphocyte activation induced an early increase of RIZ2 and RIZ1 followed by BLIMP1b increase and finally by BLIMP1a increase. The “first” and the “second” signals shifted the balance towards the PR- forms for both genes. Interestingly, the PI3K signaling pathway modulated the RIZ1/RIZ2 ratio in favor of RIZ1 while the balance versus RIZ2 was promoted by MAPK pathway. Cytokines mediating different Jak/Stat signaling pathways (third signal) early modulated the expression of PRDM1 and PRDM2 and the relationship of their different transcripts confirming the early increase of the PR- transcripts. Different responses of T cell subpopulations were also observed. Jurkat cells showed that the acute transient RIZ2 increase promoted the balancing of PRDM1 forms towards BLIMP1b. The stable forced expression of RIZ1 or RIZ2 induced a significant variation in the expression of key transcription factors involved in T lymphocyte differentiation. The BLIMP1a/b balance shifted in favor of BLIMP1a in RIZ1-overexpressing cells and of BLIMP1b in RIZ2-overexpressing cells.ConclusionsThis study provides the first characterization of PRDM2 in T-lymphocyte activation/differentiation and novel insights on PRDM1 and PRDM2 transcription regulation during initial activation phases.

Beyond Antioxidant Activity: Redox Properties of Catechins May Affect Changes in the DNA Methylation Profile-The Example of SRXN1 Gene.

The role of catechins in the epigenetic regulation of gene expression has been widely studied; however, if and how this phenomenon relates to the redox properties of these polyphenols remains unknown. Our earlier study demonstrated that exposure of the human colon adenocarcinoma HT29 cell line to these antioxidants affects the expression of redox-related genes. In particular, treatment with (-)-epigallocatechin (EGC) downregulated transcription of gene encoding sulfiredoxin-1 (SRXN1), the peroxidase involved in the protection of cells against hydrogen peroxide-induced oxidative stress. The aim of this study was to investigate whether the observed SRXN1 downregulation was accompanied by changes in the DNA methylation level of its promoter and, if so, whether it was correlated with the redox properties of catechins. The impact on DNA methylation profile in HT29 cells treated with different concentrations of five catechins, varying in chemical structures and standard reduction potentials as well as susceptibility to oxidation, was monitored by a methylation-sensitive high-resolution melting technique employing the SRXN1 promoter region as a model target. We demonstrated that catechins, indeed, are able to modulate DNA methylation of the SRXN1 gene in a redox-related manner. The nonlinear method in the statistical analysis made it possible to fish out two parameters (charge transfer in oxidation process Qox and time of electron transfer t), whose strong interactions correlated with observed modulation of DNA methylation by catechins. Based on these findings, we present a proof-of-concept that DNA methylation, which limits SRXN1 expression and thus restricts the multidirectional antioxidant action of SRXN1, may represent a mechanism protecting cells against reductive stress caused by particularly fast-reacting reductants such as EGC and (-)-epicatechin gallate (ECG) in our study.

The Role of PRMT5 in Immuno-Oncology.

Immune checkpoint inhibitor (ICI) therapy has caused a paradigm shift in cancer therapeutic strategy. However, this therapy only benefits a subset of patients. The difference in responses to ICIs is believed to be dependent on cancer type and its tumor microenvironment (TME). The TME is favorable for cancer progression and metastasis and can also help cancer cells to evade immune attacks. To improve the response to ICIs, it is crucial to understand the mechanism of how the TME is maintained. Protein arginine methyltransferase 5 (PRMT5) di-methylates arginine residues in its substrates and has essential roles in the epigenetic regulation of gene expression, signal transduction, and the fidelity of mRNA splicing. Through these functions, PRMT5 can support cancer cell immune evasion. PRMT5 is necessary for regulatory T cell (Treg) functions and promotes cancer stemness and the epithelial-mesenchymal transition. Specific factors in the TME can help recruit Tregs, tumor-associated macrophages, and myeloid-derived suppressor cells into tumors. In addition, PRMT5 suppresses antigen presentation and the production of interferon and chemokines, which are necessary to recruit T cells into tumors. Overall, PRMT5 supports an immunosuppressive TME. Therefore, PRMT5 inhibition would help recover the immune cycle and enable the immune system-mediated elimination of cancer cells.