Differentially Methylated Genes Research Articles

Integrative DNA methylome and transcriptome analysis illuminate leaf phenotype alterations in tetraploid citrus

Whole-genome duplication (WGD) in plants triggers profound morphological and physiological changes, with DNA modification being a key epigenetic factor that helps neo-polyploids overcome challenges and gain adaptive advantages. Tetraploids were previously mined from diploid citrus seedlings, showing enhanced environmental adaptability and potential as rootstocks. These tetraploids exhibited increased leaf and cell wall thickness compared to their diploid counterparts. To explore the impact of WGD, transcriptomic and whole-genome bisulfite sequencing (WGBS) were conducted on two pairs of citrus tetraploids and their diploid controls, revealing significant molecular changes. Notably, tetraploid citrus displayed lower CG methylation levels in gene and transposable element (TE) bodies relative to diploids. Differentially methylated genes (DMGs) between tetraploids and diploids were primarily associated with immune stress, organ development, metabolic pathways, and secondary metabolism. In Trifoliate orange (Poncirus trifoliata L. Raf.) and Ziyang Xiangcheng (Citrus junos Sieb. ex Tanaka), only 150 and 58 differentially expressed genes (DEGs) were identified, respectively, with enrichment in critical cellular processes such as cell wall synthesis, plastid development, and pathways modulating chloroplasts and plasma membranes. A total of 70 genes showed both differential methylation and expression, including ACN1, Nac036, and ASMT1, which are involved in stomatal development, leaf morphology, and melatonin synthesis, respectively, offering insight into the regulatory mechanisms of phenotype alterations after polyploidization. These findings reveal the epigenetic modifications in polyploid citrus and highlight the role of polyploidization-induced methylation in driving phenotypic changes.

Genome-wide methylation and transcriptome differential analysis of skeletal muscle in broilers with valgus-varus deformity

ABSTRACT 1. Valgus-varus deformity (VVD) is a disease that severely affects leg function in broilers and for which there is no effective control method current available. Although DNA methylation has an important impact on most physiological and pathological processes, its involvement in skeletal muscle growth and development in VVD broilers is unknown. In this study, genome-wide DNA methylation was analysed in VVD-affected and normal broilers using whole genome resulphite sequencing. 2. The results showed that in the cytosine-phosphoric acid-guanine (CG) sequence environment there was a methylation rate of about 55% and 4,265 differentially methylated regions (DMRs) were found in the CG. Of these, 550 were located in the promoter, 547 in the exon region, and 1,718 in the intron region. 3. All differentially methylated genes (DMGs) were analysed for enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The GO was enriched in pathways related to protein degradation such as proteasome complex, endopeptidase complex and extracellular region. The KEGG pathways were enriched in signalling pathways related to protein degradation and catabolism such as proteasome, nitrogen metabolism, adherens junction and alanine. 4. Protein interactions analysis revealed that FOS, MYL9, and FRAS1 had a high degree of interactions, in which the DNA methylation level of the MYL9 promoter region was negatively correlated with mRNA expression level. Further studies showed that 5-azacytidine (5-AzaC) inhibited DNMT1 and DNMT3A gene expression and promoted MYL9 expression. 5. This study systematically investigated overall DNA methylation patterns in the leg muscle of VVD and normal broilers. It screened common differential genes in conjunction with transcriptomic data to further identify genes associated with muscle growth and development. This study provides new insights to better understand the pathogenesis of VVD from an epigenetic perspective.

Epigenetic insights into creep-feeding: methylation profiling of Longissimus thoracis muscle at weaning in crossbred cattle

IntroductionThis study investigated the impact of creep-feeding supplementation on the genome methylation of the Longissimus thoracis (LT) muscle in crossbred beef cattle (Bos taurus × Bos indicus).MethodsThe experiment involved 48 uncastrated F1 Angus-Nellore males (half-siblings), which were divided into two groups: NCF – no creep-feeding (n = 24) and CF – creep-feeding (n = 24). After weaning at 210 days, all animals were feedlot finished for 180 days under identical conditions. LT muscle biopsies were collected at weaning for genomic DNA methylation analysis by reduced representation bisulfite sequencing (RRBS).Results and discussionThe groups differed significantly (CF > NCF: p < 0.05) to weaning weight (243.57±5.70 vs. 228.92±5.07kg), backfat thickness (12.96±0.86 vs. 10.61±0.42mm), LT muscle marbling score (366.11±12.39 vs. 321.50±13.65), and LT intramuscular fat content (5.80±0.23 vs. 4.95±0.20%). The weights at the beginning of the experiment and at slaughter (390 days) did not differ significantly. Mean methylation levels were higher in CF with 0.18% more CpG, 0.04% CHG, and 0.03% CHH. We identified 974 regions with differential methylation (DMRs: > 25% and q < 0.05), which overlapped with 241 differentially methylated genes (DMGs). Among these genes, 108 were hypermethylated and 133 were hypomethylated in CF group. Notably, 39 of these DMGs were previously identified as differentially expressed genes (DEGs: log2 fold change [0.5]) in the same animal groups. Over-representation analysis highlighted epigenetic regulations related to muscle growth, PPAR signaling, adipogenesis, insulin response, and lipid metabolism. Key DMGs/DEGs included: ACAA1, SORBS1, SMAD3, TRIM63, PRKCA, DNMT3A, RUNX1, NRG3, and SLC2A8. These epigenetic changes improved the performance of supplemented animals up to weaning and enhanced meat quality traits, particularly higher intramuscular fat. The results provided insights into the intricate interplay between nutrition, epigenetics, gene expression and phenotypes in beef cattle production.

5hmC-profiles in Puerto Rican Hispanic/Latino men with aggressive prostate cancer.

Puerto Rican (PR) Hispanic/Latino (H/L) men are an understudied population that has the highest prostate cancer (PCa) specific mortality among other Hispanic populations. Little information is known about the higher mortality in PR H/L men. It is thought that epigenetic changes in key genes may play a critical role in aggressive tumors. We aimed to identify key 5-hydroxymethylcytosine (5hmC) changes in PR H/L men with aggressive PCa. We performed sequencing analysis using the 5hmC-enriched DNA from 22 prostate tumors and 24 adjacent normal FFPE samples. We identified 808 differentially methylated genes (DMGs) in tumors compared to adjacent normal tissues (FDR<0.05, log2FC>|0.4|). Pathway analysis of DMGs demonstrated that DNA repair pathway was most upregulated in tumors. Since 5hmC abundance positively correlates with gene expression levels, we further investigated 808 DMGs in TCGA PCa gene expression data. Further, we identified 59 DMGs (80.1%, FDR<0.05, ΔGE (gene expression) >|1|) with significant gene expression changes in the same direction. Additionally, we identified 111 aggressiveness-related DMGs, of which, two hypomethylated genes ( CCDC122 , NUDT15 ) and four hypermethylated genes ( PVT1 , RPL30 , TRMT12 , UBR5 ) were found to be altered at transcriptomic level in a concordant manner in PR H/L PCa patients (N=86). The aberrant 5hmC (N=55) and GE (N=497) changes in these six genes were also associated with progression-free survival in the mixed PCa population. In conclusion, our study identified 59 DMGs showing concordant epigenetic and transcriptomic changes in tumor tissues and 111 DMGs showing association with aggressive PCa among PR H/L men.

Gender-Specific DNA Methylation Profiles Associated with Adult Weight in Hezuo Pigs.

The Hezuo pig, an important native Tibetan breed in China, exhibits differences in adult body weight, with females typically heavier than males. The underlying mechanisms for this disparity remain unclear. DNA methylation changes are known to influence animal growth and development and regulate Hezuo pig growth by altering gene expression related to these processes, thus differentially affecting adult body weight between genders. This study conducted DNA methylation analysis and expression profiling using pituitary tissues from male and female Hezuo pigs at 3 and 8 months old (M3M, M3F, M8M, and M8F). In total 346, 795, 371, and 839 differentially methylated genes (DMGs) were identified in the M3M vs. M3F, M3F vs. M8F, M3M vs. M8M, and M8M vs. M8F groups, respectively. The comparative analysis of differentially methylated regions (DMRs) genes and DEGs (differentially expressed regions) revealed that key genes involved in growth, hormone secretion, and the hypothalamic-pituitary-gonadal axis are primarily enriched in signaling pathways such as PI3K-Akt, Hippo, and adrenergic. Further analysis combining methylation and transcriptomics identified five candidate methylated genes (CCL2, MYL2, GST, CTSH, and MCH) linked to adult body weight in Hezuo pigs. Additionally, the correlation analysis suggested that these genes influence growth and development in boars and sows by regulating the secretion and synthesis of related hormones, leading to heavier weights in females. In conclusion, variations in adult body weight between male and female pigs may stem from the impact of DNA methylation on gene expression related to growth and development. These findings offer new insights into the regulatory mechanisms of DNA methylation during weight gain in Hezuo pigs.

Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock “Pd1” (Prunus tomentosa) on sweet cherry ‘Shuguang2’ scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock–scion interactions.

CHH hypermethylation contributes to the early ripening of grapes revealed by DNA methylome landscape of “Kyoho” and its bud mutant

Abstract DNA methylation is a stable epigenetic mark that plays a crucial role in plant life processes. However, the specific functions of DNA methylation in grape berry development remain largely unknown. In this study, we performed whole-genome bisulfite sequencing on “Kyoho” grape and its early-ripening bud mutant “Fengzao” at different developmental stages. Our results revealed that transposons (TEs) and gene flanking regions exhibited high levels of methylation, particularly in “Fengzao”, attributed to CHH site methylation. Interestingly, the methylation patterns in these two cultivars showed distinct dynamics during berry development. While methylation levels of genes and TEs increased gradually in “Kyoho” throughout berry development, “Fengzao” did not display consistent changes. Notably, “Fengzao” exhibited higher methylation levels in promoters compared to “Kyoho”, suggesting that hypermethylation of promoters may contribute to its early ripening phenotype. Integration of methylome and transcriptome data highlighted differentially methylated genes (DMGs) and expressed genes (DEGs) associated with secondary metabolite biosynthesis, with 38 genes identified as potential candidates involved in grape berry development. Furthermore, the study identified a jasmonate-induced oxygenase gene (JOX1) as a negative regulator of ripening in Arabidopsis and grapes, indicating that hypermethylation of JOX1 may play a role in the early ripening of “Fengzao”. Overall, our findings provide insights into the distinct DNA methylation patterns during grape berry development, shedding light on the epigenetic regulatory mechanisms underlying the early-ripening bud mutant.

Multi-omics analysis of sea urchin (Strongylocentrotus intermedius) adaptation to salt stress reveals epigenetic regulatory mechanisms

Increased precipitation and surface runoff due to global warming has reduced surface seawater and nearshore water salinity, affecting marine organism survival. Nevertheless, the epigenetic mechanisms underlying the adaptation of sea urchins (Strongylocentrotus intermedius) to low-salinity stress remains unknown. This study utilized response surface methodology to formulate growth models for S. intermedius at different salinity levels and shell diameters. Whole genome bisulphite sequencing, RNA-sequencing, and metabolome analysis were performed on low-salinity stressed sea urchins, and a total of 3006 differential methylated regions, 945 differential methylated genes, 886 differentially expressed genes, and 271 differential metabolites were identified. Multi-omics analysis revealed significant alterations in the gene expression and DNA methylation of key genes (e.g., SPHK2, Arpc1a, and SLC23A1) in S. intermedius under low-salinity stress conditions. These changes subsequently influenced the metabolism of substances, including sphingosine-1-phosphate, ethanolamine phosphate, vitamin C, taurine, and choline. These alterations impacted functions related to immunity, osmoregulation, and energy metabolism in the organism. This study was to comprehensively elucidate the epigenetic mechanisms of S. intermedius under low-salinity stress from the perspective of DNA-RNA-metabolites, providing new insights into salinity adaptation of marine invertebrates.

N6-Methyladenosine RNA Modification Regulates Maize Resistance to Maize Chlorotic Mottle Virus Infection.

Maize chlorotic mottle virus (MCMV) is one of the main viruses causing significant losses in maize. N6-methyladenosine (m6A) RNA modification has been proven to play important regulatory roles in plant development and stress response. In this study, we found that MCMV infection significantly up-regulated the m6A level in maize, and methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were performed to investigate the distribution of m6A modified peaks and gene expression patterns in MCMV-infected maize plants. The results showed that 1325 differentially methylated genes (DMGs) and 47 differentially methylated and expressed genes (DMEGs) were identified and analyzed. Moreover, the results of virus-induced gene silencing (VIGS) assays showed that ZmECT18 and ZmGST31 were required for MCMV infection, while silencing of ZmMTC, ZmSCI1 or ZmTIP1 significantly promoted MCMV infection in maize. Our findings provided novel insights into the regulatory roles of m6A modification in maize response to MCMV infection.

DNA Methylation of KLRC1 and KLRC3 in Autoimmune Thyroiditis: Perspective of Different Water Iodine Exposure

ObjectiveThis study aimed to identify differentially methylated genes (DMGs) associated with natural killer cells in patients with autoimmune thyroiditis (AIT), focusing on the influence of varying water iodine exposure levels. MethodsParticipants were divided into categories based on median water iodine (MWI) concentrations: iodine-fortified areas (IFA, MWI < 10 μg/L), iodine-adequate areas (IAA, 40 ≤ MWI ≤ 100 μg/L), and iodine-excessive areas (IEA, MWI > 300 μg/L). A total of 176 matched AIT cases and controls were recruited and divided into 89, 40, and 47 pairs for IFA, IAA, and IEA, respectively. DMGs were identified using 850K BeadChip analysis for 10/10 paired samples. Validation of DNA methylation and mRNA expression levels of the DMGs was conducted using MethylTarget™ and QRT-PCR for 176/176 paired samples. ResultsKLRC1, KLRC3, and SH2D1B were identified as significant DMGs. Validation revealed that KLRC1 was hypomethylated and highly expressed, whereas KLRC3 was hypermethylated and highly expressed in individuals with AIT. Furthermore, KLRC1 was hypomethylated and highly expressed in both IFA and IEA. ConclusionThe DNA methylation status of KLRC1 and KLRC3 may play crucial roles in AIT pathogenesis. Additionally, DNA methylation of KLRC1 seems to be influenced by different iodine concentrations in water.

438 DNA methylation patterns and transcriptional regulation during pig fetal skeletal muscle development

Abstract Fetal development is controlled by a complex cascade of highly regulated and coordinated gene expression patterns. Epigenetic mechanisms have important roles in regulating development and differentiation. Among such mechanisms, DNA methylation exhibits context-specific associations with gene expression and has been shown to be highly dynamic during developmental processes. We performed whole-genome bisulfite sequencing (WGBS) to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-d gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45,739 differentially methylated regions (DMRs) between stages, and the majority (n = 34,232) were hypomethylated at 70 vs. 41 dg. Developmental DMRs exhibited feature-specific enrichment in gene regulatory regions, as well as in regions proximal to micro-RNAs (miRNAs) that have known roles in myogenesis. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and transcript abundance. We surveyed myogenic regulatory factor (MRF) genes to determine if differential methylation was present in expected genomic regions. Within the MYF5 and MYF6 locus, MYF5 was significantly promoter-hypermethylated at 70 dg, whereas MYF6 was significantly hypomethylated upstream of its transcription start site. MYF5 is the earliest MRF to be expressed and primarily functions in myoblast proliferation and determination, while MYF6 functions in muscle cell differentiation. Thus, these patterns were consistent with expected downregulation of MYF5 and upregulation of MYF6 as muscle development progresses and demonstrate that differential methylation is evident at myogenic transcription factors. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We also show that developmental DMRs are enriched for GWAS SNPs associated with muscle physiology and meat quality traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics in pig fetal skeletal muscle and reveal putative cis-regulatory elements governed by epigenetic processes during porcine myogenesis.

Comprehensive Analysis of Methylome and Transcriptome to Identify Potential Genes Regulating Porcine Testis Development.

DNA methylation plays a critical role in regulating gene expression during testicular development. However, few studies report on candidate genes related to the DNA methylation regulation of porcine testicular development. This study examined the differentially expressed genes (DEGs) and their methylation levels in testicular tissues from pigs at 60 days of age (60 d) and 180 days of age (180 d) using RNA-Seq and whole genome bisulfite sequencing (WGBS). It was determined that DNA methylation primarily occurs in the cytosine-guanine (CG) context, and the analysis identified 106,282 differentially methylated regions (DMRs) corresponding to 12,385 differentially methylated genes (DMGs). Further integrated analysis of RNA-Seq and WGBS data revealed 1083 DMGs negatively correlated with the expression of DEGs. GO analysis showed that these genes were significantly enriched in spermatogenesis, germ cell development, and spermatid differentiation. The screening of enriched genes revealed that hyper-methylation repressed ADAM30, ADAM3A, DPY19L2, H2BC1, MAK, RPL10L, SPATA16, and YBX2, while hypo-methylation elevated CACNA1I, CADM1, CTNNB1, JAM2, and PAFAH1B3 expression. Additionally, the methylation status of the key genes ADAM3A, ADAM30, YBX2, JAM2, PAFAH1B3, and CTNNB1 was detected by bisulfite sequencing PCR (BSP). This study offers insights into the epigenetic regulation mechanisms underlying porcine testicular development.

Methylation-related differentially expressed genes as potential prognostic biomarkers for cervical cancer

AimTo discover novel methylation-related differentially expressed genes (MRDEGs) for cervical cancer, with a focus on their potential for early diagnosis and prognostic assessment. Materials & methodsWe integrated data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. TCGA-MRDEGs were identified by analyzing differentially methylated genes (DMGs) and their correlation with gene expression. We examined GEO datasets GSE39001, GSE9750, and GSE46306 for GEO-MRDEGs. Overlapping MRDEGs were subjected to overall survival (OS) analysis to identify prognostic markers. The expression and methylation levels of these genes were validated in a total of 30 tissue samples, comprising 20 from cervical cancer patients and 10 from normal cervical tissues, using qRT-PCR and MassARRAY EpiTYPER Assay. ResultsA total of 314 TCGA-MRDEGs and 40 GEO-MRDEGs were identified. Intersection analysis yielded 10 overlapping MRDEGs. Notably, NOVA1, GSTM5, TRHDE, and CXCL12 were found to have reduced expression and increased methylation in cervical cancer, which correlated with poor prognosis. The methylation status and expression levels of these genes were confirmed in tissue specimens. ConclusionWe identified four MRDEGs as potential prognostic biomarkers for cervical cancer. Their clinical utility is highlighted, but further validation in larger cohorts is required to establish their clinical significance.

Genome-wide methylation and gene-expression analyses in thalassemia.

Thalassemia is the most common autosomal genetic disorder in humans. The pathogenesis of thalassemia is principally due to the deletion or mutation of globin genes that then leads to disorders in globin-chain synthesis, and its predominant clinical manifestations include chronic forms of hemolytic anemia. However, research on the epigenetics and underlying pathogenesis of thalassemia is in its nascency and not yet been systematically realized. In this study, we compared the results of RNA-seq and the whole-genome bisulfite sequencing (WGBS) on 22 peripheral blood samples from 14 thalassemic patients and eight healthy individuals revealed a genome-wide methylation landscape of differentially methylated regions (DMRs). And functional-enrichment analysis revealed the enriched biological pathways with respect to the differentially expressed genes (DEGs) and differentially methylated genes (DMGs) to include hematopoietic lineage, glucose metabolism, and ribosome. To further analyze the interaction between the transcriptome and methylome, we implemented a comprehensive analysis of overlaps between DEGs and DMGs, and observed that biological processes significantly enriched the immune-related genes (i.e., our hypermethylated and down-regulated gene group). Hypermethylated and hypomethylated regions of thalassemia-related genes exhibited different distribution patterns. We thus, further identified and validated thalassemia-associated DMGs and DEGs by multi-omics integrative analyses of DNA methylation and transcriptomics data, and provided a comprehensive genomic map of thalassemia that will facilitate the exploration of the epigenetics mechanisms and pathogenesis underlying thalassemia.

Pesticide-induced transgenerational alterations of genome-wide DNA methylation patterns in the pancreas of Xenopus tropicalis correlate with metabolic phenotypes

The unsustainable use of manmade chemicals poses significant threats to biodiversity and human health. Emerging evidence highlights the potential of certain chemicals to cause transgenerational impacts on metabolic health. Here, we investigate male transmitted epigenetic transgenerational effects of the anti-androgenic herbicide linuron in the pancreas of Xenopus tropicalis frogs, and their association with metabolic phenotypes. Reduced representation bisulfite sequencing (RRBS) was used to assess genome-wide DNA methylation patterns in the pancreas of adult male F2 generation ancestrally exposed to environmentally relevant linuron levels (44 ± 4.7 μg/L). We identified 1117 differentially methylated regions (DMRs) distributed across the X. tropicalis genome, revealing potential regulatory mechanisms underlying metabolic disturbances. DMRs were identified in genes crucial for pancreatic function, including calcium signalling (clstn2, cacna1d and cadps2), genes associated with type 2 diabetes (tcf7l2 and adcy5) and a biomarker for pancreatic ductal adenocarcinoma (plec). Correlation analysis revealed associations between DNA methylation levels in these genes and metabolic phenotypes, indicating epigenetic regulation of glucose metabolism. Moreover, differential methylation in genes related to histone modifications suggests alterations in the epigenetic machinery. These findings underscore the long-term consequences of environmental contamination on pancreatic function and raise concerns about the health risks associated with transgenerational effects of pesticides.

Epigenome-wide differential methylation and differential variability as predictors of high-grade cervical intraepithelial neoplasia (cin2+).

CpG site methylation patterns have potential to improve differentiation of high-grade screening-detected cervical abnormalities. We assessed CpG differential methylation (DM) and differential variability (DV) in high-grade (CIN2+) vs. low-grade (≤CIN1) lesions. In ≤CIN1 (n=117) and CIN2+ (n=31) samples, cervical sample DNA underwent testing with Illumina HumanMethylation arrays. We assessed DM and DV of CpG methylation M values among nine cervical cancer-associated genes. We fit CpG-specific linear models and estimated empirical Bayes standard errors and false discovery rates (FDR). An exploratory epigenome-wide association study (EWAS) aimed to detect novel DM and DV CpGs (FDR<0.05) and Gene Ontology (GO) term enrichment. Compared to ≤CIN1, CIN2+ exhibited greater methylation at CCNA1 Cluster 1 (M value difference 0.24; 95% CI 0.04, 0.43) and RARB Cluster 2 (0.16; 95% CI 0.05, 0.28), and lower methylation at CDH1 Cluster 1 (-0.15; 95% CI -0.26, -0.04). CIN2+ exhibited lower variability at CDH1 Cluster 2 (variation difference -0.24; 95% CI -0.41, -0.05) and FHIT Cluster 1 (-0.30; 95% CI -0.50, -0.09). EWAS detected 3,534 DM and 270 DV CpGs. Forty-four GO terms were enriched with DM CpGs related to transcriptional, structural, developmental, and neuronal processes. Methylation patterns may help triage screening-detected cervical abnormalities and inform US screening algorithms.

Extensive differential DNA methylation between tuberculosis skin test positive and skin test negative cattle

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), represents a significant problem for the agriculture industry as well as posing a risk for human health. Current diagnostic tests for bTB target the cell-mediated immune (CMI) response to infection with M. bovis, primarily through screening of animals with the tuberculin skin test. Epigenetic modifications have been shown to alter the course of the immune response and differentially methylated regions (DMRs) might also influence the outcome of the skin test in cattle. Whole Genome Bisulphite Sequencing (WGBS) was used to profile DNA methylation levels from peripheral blood of a group of cattle identified as test positive for M. bovis (positive for the single intradermal comparative tuberculin test (SICTT) and/or the interferon-γ release assay compared to a test negative control group [n = 8/group, total of 16 WGBS libraries]. Although global methylation profiles were similar for both groups across the genome, 223 DMRs and 159 Differentially Promoter Methylated Genes (DPMGs) were identified between groups with an excess of hypermethylated sites in SICTT positive cattle (threshold > 15% differential methylation). Genes located within these DMRs included the Interleukin 1 receptor (IL1R1) and MHC related genes (BOLA and BOLA-DQB). KEGG pathway analysis identified enrichment of genes involved in Calcium and MAPK signalling, as well as metabolism pathways. Analysis of DMRs in a subset of SICTT negative cattle that were IFN-γ positive showed differential methylation of genes including Interleukin 10 Receptor, alpha (IL10RA), Interleukin 17 F (IL17F) and host defence peptides (DEFB and BDEF109). This study has identified a number of immune gene loci at which differential methylation is associated with SICTT test results and the degree of methylation could influence effective host immune responses.

Comprehensive analysis of aberrantly methylated differentially expressed genes and validation of CDC6 in melanoma

BackgroundSkin Cutaneous Melanoma (SKCM) is a highly aggressive malignant tumor with a significant increase in mortality upon metastasis. The molecular mechanisms driving melanoma progression remain largely unclear. Recent studies have highlighted the importance of epigenetic alterations, especially DNA methylation, in melanoma development. This study aims to identify and analyze methylation-regulated differentially expressed genes (MeDEGs) in genome-wide profiles between primary and metastatic melanoma.MethodsGene expression profiling datasets GSE8401 and gene methylation profiling datasets GSE86355 were collected from the GEO database. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were systematically identified. Integration of DEGs and DMGs yielded a set of MeDEGs, which subsequently underwent functional enrichment analysis. The protein-protein interaction (PPI) network was constructed using STRING and visualized using Cytoscape software. Survival analysis was used to select prognostic hub genes. In addition, 37 SKCM and 37 normal skin tissues from the First Affiliated Hospital of Soochow University (FAHSU) were collected for immunohistochemical (IHC) staining and evaluation. Furthermore, DNA methylation patterns of CDC6 were analyzed. To validate these findings, SKCM cell cultures were utilized to elucidate the expression and behavioral characteristics of CDC6. Additionally, gene set enrichment analysis (GSEA) and immune infiltration analysis were conducted for CDC6.ResultsIn our study, we discovered 120 hypomethylated-upregulated genes and 212 hypermethylated-downregulated genes. The hypomethylated-upregulated genes were notably associated with biological processes such as spindle assembly checkpoint signaling, mitotic spindle assembly, and negative regulation of mitotic metaphase/anaphase transition. Our pathway analysis revealed significant enrichment in pathways related to dilated cardiomyopathy, amino sugar metabolism, progesterone-mediated oocyte maturation, and chemical carcinogenesis. Conversely, hypermethylated-downregulated genes were found to be enriched in processes like epidermis development, keratinocyte differentiation, and skin development. Additionally, pathway analysis highlighted associations with estrogen signaling, Staphylococcus aureus infection, axon guidance, and arachidonic acid metabolism. Following the establishment of PPI networks and survival analysis, we identified 11 prognostic hub genes: CCNA2, CDC6, CDCA3, CKS2, DTL, HJURP, KRT5, KRT14, KRT15, KRT16, and NEK2. Notably, among the 11 hub genes, our findings indicate that CDC6 plays a pivotal role in enhancing the proliferation, migration, and invasion capabilities of melanoma cells in vitro.ConclusionsOur comprehensive genomic analyses reveal that genes with aberrant methylation exhibit differential expression during the transition from primary to metastatic melanoma. The identified genes, especially CDC6, which plays a crucial role in enhancing melanoma cell proliferation, migration, and invasion, provide valuable insights into potential methylation-based biomarkers. These findings could contribute significantly to advancing precision medicine in SKCM.

DNA Methylation Profiling in Genetically Selected Clarias magur (Hamilton, 1822) Provides Insights into the Epigenetic Regulation of Growth and Development.

DNA methylation is an epigenetic alteration that impacts gene expression without changing the DNA sequence affecting an organism's phenotype. This study utilized a reduced representation bisulfite sequencing (RRBS) approach to investigate the patterns of DNA methylation in genetically selected Clarias magur stocks. RRBS generated 249.22 million reads, with an average of 490,120 methylation sites detected in various parts of genes, including exons, introns, and intergenic regions. A total of 896 differentially methylated regions (DMRs) were identified; 356 and 540 were detected as hyper-methylated and hypo-methylated regions, respectively. The DMRs and their association with overlapping genes were explored using whole genome data of magur, which revealed 205 genes in exonic, 210 in intronic, and 480 in intergenic regions. The analysis identified the maximum number of genes enriched in biological processes such as RNA biosynthetic process, response to growth factors, nervous system development, neurogenesis, and anatomical structure morphogenesis. Differentially methylated genes (DMGs) such as myrip, mylk3, mafb, egr3, ndnf, meis2a, foxn3, bmp1a, plxna3, fgf6, sipa1l1, mcu, cnot8, trim55b, and myof were associated with growth and development. The selected DMGs were analyzed using real-time PCR, which showed altered mRNA expression levels. This work offers insights into the epigenetic mechanisms governing growth performance regulation in magur stocks. This work provides a valuable resource of epigenetic data that could be integrated into breeding programs to select high-performing individuals.

Transcriptomic and Epigenomic Responses to Cortisol-Mediated Stress in Rainbow Trout (Oncorhynchus mykiss) Skeletal Muscle.

The production and release of cortisol during stress responses are key regulators of growth in teleosts. Understanding the molecular responses to cortisol is crucial for the sustainable farming of rainbow trout (Oncorhynchus mykiss) and other salmonid species. While several studies have explored the genomic and non-genomic impacts of cortisol on fish growth and skeletal muscle development, the long-term effects driven by epigenetic mechanisms, such as cortisol-induced DNA methylation, remain unexplored. In this study, we analyzed the transcriptome and genome-wide DNA methylation in the skeletal muscle of rainbow trout seven days after cortisol administration. We identified 550 differentially expressed genes (DEGs) by RNA-seq and 9059 differentially methylated genes (DMGs) via whole-genome bisulfite sequencing (WGBS) analysis. KEGG enrichment analysis showed that cortisol modulates the differential expression of genes associated with nucleotide metabolism, ECM-receptor interaction, and the regulation of actin cytoskeleton pathways. Similarly, cortisol induced the differential methylation of genes associated with focal adhesion, adrenergic signaling in cardiomyocytes, and Wnt signaling. Through integrative analyses, we determined that 126 genes showed a negative correlation between up-regulated expression and down-regulated methylation. KEGG enrichment analysis of these genes indicated participation in ECM-receptor interaction, regulation of actin cytoskeleton, and focal adhesion. Using RT-qPCR, we confirmed the differential expression of lamb3, itga6, limk2, itgb4, capn2, and thbs1. This study revealed for the first time the molecular responses of skeletal muscle to cortisol at the transcriptomic and whole-genome DNA methylation levels in rainbow trout.