Maternal Undernutrition Affects Fetal Thymus DNA Methylation, Gene Expression, and, Thereby, Metabolism and Immunopoiesis in Wagyu (Japanese Black) Cattle.

We aimed to determine the effects of maternal nutrient restriction (MNR) on the DNA methylation and gene expression patterns associated with metabolism and immunopoiesis in the thymuses of fetal Wagyu cattle. Pregnant cows were allocated to two groups: a low-nutrition (LN; 60% nutritional requirement; n = 5) and a high-nutrition (HN; 120% nutritional requirement, n = 6) group, until 8.5 months of gestation. Whole-genome bisulfite sequencing (WGBS) and RNA sequencing were used to analyze DNA methylation and gene expression, while capillary electrophoresis-Fourier transform mass spectrometry assessed the metabolome. WGBS identified 4566 hypomethylated and 4303 hypermethylated genes in the LN group, with the intergenic regions most frequently being methylated. Pathway analysis linked hypoDMGs to Ras signaling, while hyperDMGs were associated with Hippo signaling. RNA sequencing found 94 differentially expressed genes (66 upregulated, 28 downregulated) in the LN group. The upregulated genes were tied to metabolic pathways and oxidative phosphorylation; the downregulated genes were linked to natural killer cell cytotoxicity. Key overlapping genes (GRIA1, CACNA1D, SCL25A4) were involved in cAMP signaling. The metabolomic analysis indicated an altered amino acid metabolism in the MNR fetuses. These findings suggest that MNR affects DNA methylation, gene expression, and the amino acid metabolism, impacting immune system regulation during fetal thymus development in Wagyu cattle.

DMAP2: A Pipeline for Analysis of Whole-Genome-Scale DNA Methylation Sequencing Data.

DNA methylation is well-established as a major epigenetic mechanism that can control gene expression and is involved in both normal development and disease. Analysis of high-throughput-sequencing-based DNA methylation data is a step toward understanding the relationship between disease and phenotype. Analysis of CpG methylation at single-base resolution is routinely done by bisulfite sequencing, in which methylated Cs remain as C while unmethylated Cs are converted to U, subsequently seen as T nucleotides. Sequence reads are aligned to the reference genome using mapping tools that accept the C-T ambiguity. Then, various statistical packages are used to identify differences in methylation between (groups of) samples. We have previously developed the Differential Methylation Analysis Pipeline (DMAP) as an efficient, fast, and flexible tool for this work, both for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS). The protocol described here includes a series of scripts that simplify the use of DMAP tools and that can accommodate the wider range of input formats now in use to perform analysis of whole-genome-scale DNA methylation sequencing data in various biological and clinical contexts. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: DMAP2 workflow for whole-genome bisulfite sequencing (WGBS) and reduced-representation bisulfite sequencing (RRBS).

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.

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.

Genomic context determines the effect of DNA methylation on gene expression in the gut epithelium of Atlantic salmon (Salmo salar).

The canonical view of DNA methylation, a pivotal epigenetic regulation mechanism in eukaryotes, dictates its role as a suppressor of gene activity, particularly within promoter regions. However, this view is being challenged as it is becoming increasingly evident that the connection between DNA methylation and gene expression varies depending on the genomic location and is therefore more complex than initially thought. We examined DNA methylation levels in the gut epithelium of Atlantic salmon (Salmo salar) using whole-genome bisulfite sequencing, which we correlated with gene expression data from RNA sequencing of the same gut tissue sample (RNA-seq). Assuming epigenetic signals might be pronounced between distinctive phenotypes, we compared large and small fish, finding 22 significant associations between 22 differentially methylated regions and 21 genes. We did not detect significant methylation differences between large and small fish. However, we observed a consistent signal of methylation levels around the transcription start sites (TSS), being negatively correlated with the expression levels of those genes. We found both negative and positive associations of methylation levels with gene expression further upstream or downstream of the TSS, revealing a more unpredictable pattern. The 21 genes showing significant methylation-expression correlations were involved in biological processes related to salmon health, such as growth and immune responses. Deciphering how DNA methylation affects the expression of such genes holds great potential for future applications. For instance, our results suggest the importance of genomic context in targeting epigenetic modifications to improve the welfare of aquaculture species like Atlantic salmon.

Discovery and Validation of Methylation Signatures in Circulating Cell-Free DNA for the Detection of Colorectal Cancer.

This study was conducted with the primary objective of assessing the performance of cfDNA methylation in the detection of colorectal cancer (CRC). Five tumor tissue, 20 peripheral blood leucocyte, and 169 cfDNA samples were collected for whole-genome bisulfite sequencing (WGBS) analysis. Bioinformatic analysis was conducted to identify differentially methylated regions (DMRs) and their functional characteristics. Quantitative methylation-specific PCR (qMSP) was used to validate the methylation levels of DMRs in the tissues and leucocytes. cfDNA samples from CRC patients and healthy controls were used to evaluate the performance of the DMR analysis. WGBS analysis revealed a decrease in DNA methylation levels in the CpG context in CRC tumor tissues compared with adjacent normal tissues. A total of 132 DMRs in cfDNA were identified as potential markers for diagnosing CRC. In a cohort of 95 CRC patients and 74 healthy controls, a combination of the three DMRs (DAB1, PPP2R5C, and FAM19A5) yielded an AUC of 0.763, achieving 64.21% sensitivity and 78.38% specificity in discriminating CRC patients from healthy controls. This study provides insights into DNA methylation patterns in CRC and identifies a set of DMRs in cfDNA with potential diagnostic value for CRC. These DMRs hold promise as biomarkers for CRC detection, offering promise for non-invasive CRC diagnosis. Further research is warranted to validate these findings in larger cohorts.

Comparing methylation levels assayed in GC-rich regions with current and emerging methods.

DNA methylation is an epigenetic mechanism that regulates gene expression, and for mammals typically occurs on cytosines within CpG dinucleotides. A significant challenge for methylation detection methods is accurately measuring methylation levels within GC-rich regions such as gene promoters, as inaccuracies compromise downstream biological interpretation of the data. To address this challenge, we compared methylation levels assayed using four different Methods Enzymatic Methyl-seq (EM-seq), whole genome bisulphite sequencing (WGBS), Infinium arrays (Illumina MethylationEPIC, "EPIC"), and Oxford Nanopore Technologies nanopore sequencing (ONT) applied to human DNA. Overall, all methods produced comparable and consistent methylation readouts across the human genome. The flexibility offered by current gold standard WGBS in interrogating genome-wide cytosines is surpassed technically by both EM-seq and ONT, as their coverages and methylation readouts are less prone to GC bias. These advantages are tempered by increased laboratory time (EM-seq) and higher complexity (ONT). We further assess the strengths and weaknesses of each method, and provide recommendations in choosing the most appropriate methylation method for specific scientific questions or translational needs.

Whole-Genome Bisulfite Sequencing Protocol for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results.

Grafting based DNA methylation alteration of snoRNAs in upland cotton (Gossypium L.).

The effects of grafting in response to various biotic and abiotic stressors have been studied, however, the methylation status of small nucleolar RNA (snoRNA) genes in heterograft and homograft cotton needs investigation. This study was undertaken to determine grafting effects on DNA methylation of snoRNA genes in Upland cotton. Rootstocks used were Pima 3-79 (Gossypium barbadense acc. Pima 3-79) and Texas Marker-1 (G. hirsutum acc. TM-1), representing two different species with different fiber properties, adaptations, and morphologies. The methylation ratio and differently methylated cytosines (DMCs) of 10935 snoRNA genes in mature seeds of heterograft and homograft cotton samples were studied using the whole genome bisulfite sequencing method. Seedling vigor and seed weight were studied to investigate phenotype alterations that might be associated with altered methylation levels among grafts. Statistically significant DMC differences among gene elements of snoRNA genes and between homograft and heterograft cotton samples were identified in the absence of DNA sequence alterations. DNA methylation alterations of snoRNA genes associated with seedling vigor and 100 seed weight. The majority of snoRNA genes showed higher numbers of mCG + mCHG-DMCs with increased methylation levels in heterograft, while there were higher numbers of mCG + mCHG-DMCs with decreased methylation levels in homograft. Since snoRNAs regulate essential genes for plant growth and development and plant adaptation to different habitats or extreme environments, their altered methylation levels should be related with plant physiology.

Whole-genome bisulfite sequencing data analysis learning module on Google Cloud Platform.

This study describes the development of a resource module that is part of a learning platform named 'NIGMS Sandbox for Cloud-based Learning' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module is designed to facilitate interactive learning of whole-genome bisulfite sequencing (WGBS) data analysis utilizing cloud-based tools in Google Cloud Platform, such as Cloud Storage, Vertex AI notebooks and Google Batch. WGBS is a powerful technique that can provide comprehensive insights into DNA methylation patterns at single cytosine resolution, essential for understanding epigenetic regulation across the genome. The designed learning module first provides step-by-step tutorials that guide learners through two main stages of WGBS data analysis, preprocessing and the identification of differentially methylated regions. And then, it provides a streamlined workflow and demonstrates how to effectively use it for large datasets given the power of cloud infrastructure. The integration of these interconnected submodules progressively deepens the user's understanding of the WGBS analysis process along with the use of cloud resources. Through this module, we can enhance the accessibility and adoption of cloud computing in epigenomic research, speeding up the advancements in the related field and beyond. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

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.

Single generation epigenetic change in captivity and reinforcement in subsequent generations in a delta smelt (Hypomesus transpacificus) conservation hatchery.

A refugial population of the endangered delta smelt (Hypomesus transpacificus) has been maintained at the Fish Conservation and Culture Laboratory (FCCL) at UC Davis since 2008. Despite intense genetic management, fitness differences between wild and cultured fish have been observed at the FCCL. To investigate the molecular underpinnings of hatchery domestication, we used whole-genome bisulfite sequencing to quantify epigenetic differences between wild and hatchery-origin delta smelt. Differentially methylated regions (DMRs) were identified from 104 individuals by comparing the methylation patterns in different generations of hatchery fish (G1, G2, G3) with their wild parents (G0). We discovered a total of 132 significant DMRs (p < .05) between G0 and G1, 132 significant DMRs between G0 and G2, and 201 significant DMRs between G0 and G3. Our results demonstrate substantial differences in methylation patterns emerged between the wild and hatchery-reared fish in the early generations in the hatchery, with a higher proportion of hypermethylated DMRs in hatchery-reared fish. The rearing environment was found to be a stronger predictor of individual clustering based on methylation patterns than family, sex or generation. Our study indicates a reinforcement of the epigenetic status with successive generations in the hatchery environment, as evidenced by an increase in methylation in hypermethylated DMRs and a decrease in methylation in hypomethylated DMRs over time. Lastly, our results demonstrated heterogeneity in inherited methylation pattern in families across generations. These insights highlight the long-term consequences of hatchery practices on the epigenetic landscape, potentially impacting wild fish populations.

High-resolution methylome analysis uncovers stress-responsive genomic hotspots and drought-sensitive transposable element superfamilies in the clonal Lombardy poplar.

DNA methylation is environment-sensitive and can mediate stress responses. In trees, changes in the environment might cumulatively shape the methylome landscape over time. However, because high-resolution methylome studies usually focus on single environmental cues, the stress-specificity and long-term stability of methylation responses remain unclear. Here, we studied the methylome plasticity of a Populus nigra cv. 'Italica' clone widely distributed across Europe. Adult trees from different geographic locations were clonally propagated in a common garden experiment and exposed to cold, heat, drought, herbivory, rust infection, and salicylic acid treatments. Whole-genome bisulfite sequencing revealed stress-induced and naturally occurring DNA methylation variants. In CG/CHG contexts, the same genomic regions were often affected by multiple stresses, suggesting a generic methylome response. Moreover, these variants showed striking overlap with naturally occurring methylation variants between trees from different locations. Drought treatment triggered CHH hypermethylation of transposable elements, affecting entire superfamilies near drought-responsive genes. Thus, we revealed genomic hotspots of methylation change that are not stress-specific and that contribute to natural DNA methylation variation, and identified stress-specific hypermethylation of entire transposon superfamilies with possible functional consequences. Our results underscore the importance of studying multiple stressors in a single experiment for recognizing general versus stress-specific methylome responses.

A strategy for studying epigenetic diversity in natural populations: proof of concept in poplar and oak.

In the last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulfite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, taking into account the structural complexity of genomes, their size, and their content in unique coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by sequencing capture bisulfite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

Genome-wide DNA methylation profiles regulate distinct heat stress response in zebu (Bos indicus) and crossbred (Bos indicus × Bos taurus) cattle.

Epigenetic variations result from long-term adaptation to environmental factors. The Bos indicus (zebu) adapted to tropical conditions, whereas Bos taurus adapted to temperate conditions; hence native zebu cattle and its crossbred (B indicus × B taurus) show differences in responses to heat stress. The present study evaluated genome-wide DNA methylation profiles of these two breeds of cattle that may explain distinct heat stress responses. Physiological responses to heat stress and estimated values of Iberia heat tolerance coefficient and Benezra's coefficient of adaptability revealed better relative thermotolerance of Hariana compared to the Vrindavani cattle. Genome-wide DNA methylation patterns were different for Hariana and Vrindavani cattle. The comparison between breeds indicated the presence of 4599 significant differentially methylated CpGs with 756 hypermethylated and 3845 hypomethylated in Hariana compared to the Vrindavani cattle. Further, we found 79 genes that showed both differential methylation and differential expression that are involved in cellular stress response functions. Differential methylations in the microRNA coding sequences also revealed their functions in heat stress responses. Taken together, epigenetic differences represent the potential regulation of long-term adaptation of Hariana (B indicus) cattle to the tropical environment and relative thermotolerance.

CelFiE-ISH: a probabilistic model for multi-cell type deconvolution from single-molecule DNA methylation haplotypes.

Deconvolution methods infer quantitative cell type estimates from bulk measurement of mixed samples including blood and tissue. DNA methylation sequencing measures multiple CpGs per read, but few existing deconvolution methods leverage this within-read information. We develop CelFiE-ISH, which extends an existing method (CelFiE) to use within-read haplotype information. CelFiE-ISH outperforms CelFiE and other existing methods, achieving 30% better accuracy and more sensitive detection of rare cell types. We also demonstrate the importance of marker selection and of tailoring markers for haplotype-aware methods. While here we use gold-standard short-read sequencing data, haplotype-aware methods will be well-suited for long-read sequencing.

The survival, gene expression, and DNA methylation of Paralichthys olivaceus impacted by the decay of green tide and bacterial infection in both laboratory and field simulation experiments.

The recurring appearance of Ulva prolifera green tides has become a pressing environmental issue, especially for marine transportation, tourism, and aquaculture in the stage of decomposition. An abundance of decaying U. prolifera leads to water acidification, hypoxia and pathogenic microorganism proliferation, threatening marine germplasm resources, particularly benthic organisms with weak escape ability. Epigenetic modification is considered to be one of the molecular mechanisms involved in the plastic adaptive response to environmental changes. However, few studies concerning the specific impact of decaying green tide on benthic animals at the epigenetic level. In this study, decomposing algal effluents of U. prolifera, sediments containing uncorrupted U. prolifera, pathogenic microorganism were considered as impact factors, to reveal the effect of decaying U. prolifera on marine economic benthic species, Paralichthys olivaceus, using both field and laboratory simulation experiments. Field simulation experiment showed higher mortality rates and serious histopathological damage than the laboratory simulation experiment. And both the decaying U. prolifera and the sediment containing U. prolifera were harmful to P. olivaceus. Genome-wide DNA methylation and transcription correlation analyses showed that the response of P. olivaceus to green tide stress and bacterial infection was mainly mediated by immune signaling pathways such as PI3K-Akt signaling pathway. DNA methylation regulates the expression of immune-related genes involved in the PI3K-Akt signaling pathway, which enables P. olivaceus to adapt to the adverse environmental stresses by resisting apoptosis. In summary, this research analyzed the potential role of P. olivaceus in decaying U. prolifera, which is of great significance for understanding the impact of decaying green tide on marine commercial fish and also provides some theoretical guidance for the proliferation and release of fish seedlings.

Integrated analysis of the DNA methylome and RNA transcriptome during the development of skeletal muscle in Duroc pigs.

BACKGROUND: Skeletal muscle development plays a crucial role in yield and quality of pork; however, this process is influenced by various factors. In this study, we employed whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing to comprehensively investigate the longissimus dorsi muscle (LDM), aiming to identify key genes that impact the growth and development of Duroc pigs with different average daily gains (ADGs). RESULTS: Eight pigs were selected and divided into two groups based on ADGs: H (774.89 g) group and L (658.77 g) group. Each pair of the H and L groups were half-siblings. The results of methylation sequencing revealed 2631 differentially methylated genes (DMGs) involved in metabolic processes, signalling, insulin secretion, and other biological activities. Furthermore, a joint analysis was conducted on these DMGs and the differentially expressed genes (DEGs) obtained from transcriptome sequencing of the same individual. This analysis identified 316 differentially methylated and differentially expressed genes (DMEGs), including 18 DMEGs in promoter regions and 294 DMEGs in gene body regions. Finally, LPAR1 and MEF2C were selected as candidate genes associated with muscle development. Bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qRT-PCR) revealed that the promoter region of LPAR1 exhibited significantly lower methylation levels (P < 0.05) and greater expression levels (P < 0.05) in the H group than in the L group. Additionally, hypermethylation was observed in the gene body region of MEF2C, as was a low expression level, in the H group (P < 0.05). CONCLUSIONS: These results suggest that the differences in the ADGs of Duroc pigs fed the same diet may be influenced by the methylation levels and expression levels of genes related to skeletal muscle development.

CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to prevent DNA hypermethylation and ensure normal transcription in growing oocytes.

The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding protein 2 (MeCP2), an epigenetic regulator displaying specifical binding with methylated DNA, remains unknown in oogenesis. In this study, we found MeCP2 protein was highly expressed in primordial and primary follicle, but was almost undetectable in secondary follicles. However, in aged ovary, MeCP2 protein is significantly increased in both oocyte and granulosa cells. Overexpression of MeCP2 in growing oocyte caused transcription dysregulation, DNA hypermethylation, and genome instability, ultimately leading to follicle growth arrest and apoptosis. MeCP2 is targeted by DCAF13, a substrate recognition adaptor of the Cullin 4-RING (CRL4) E3 ligase, and polyubiquitinated for degradation in both cells and oocytes. Dcaf13-null oocyte exhibited an accumulation of MeCP2 protein, and the partial rescue of follicle growth arrest induced by Dcaf13 deletion was observed following MeCP2 knockdown. The RNA-seq results revealed that large amounts of genes were regulated by the DCAF13-MeCP2 axis in growing oocytes. Our study demonstrated that CRL4DCAF13 E3 ubiquitin ligase targets MeCP2 for degradation to ensure normal DNA methylome and transcription in growing oocytes. Moreover, in aged ovarian follicles, deceased DCAF13 and DDB1 protein were observed, indicating a potential novel mechanism that regulates ovary aging.

Genome-wide DNA methylation changes in Oryzias melastigma embryos exposed to the water accommodated fraction of crude oil.

The water accommodated fraction (WAF) of crude oil exerts considerable impacts on marine fish during embryonic stage. Clarifying changes in epigenetic modifications is helpful for understanding the molecular mechanism underlying the toxicity of embryonic WAF exposure. The aim of this study was to explore genome-wide DNA methylation changes in Oryzias melastigma embryos after exposure to the nominal total petroleum hydrocarbon concentration of 500 µg/L in WAF for 7 days. Whole-genome bisulfite sequencing revealed that 8.47 % and 8.46 % of all the genomic C sites were methylated in the control and WAF-exposed groups, respectively. Among the three sequence contexts, methylated CG site had the largest number in both the two groups. The sequence preferences of nearby methylated cytosines were consistent between the two groups. A total of 4798 differentially methylated regions (DMRs) were identified in the promoter region. Furthermore, Gene Ontology analysis revealed that DMR-related genes were enriched mainly for functions related to development and nervous system. Additionally, the Kyoto Encyclopedia of Genes and Genomes pathways enriched in DMR-related genes were related to nervous system and endocrine system. These novel findings provide comprehensive insights into the genome-wide DNA methylation landscape of O. melastigma following embryonic WAF exposure, shedding light on the epigenetic regulatory mechanisms underlying WAF-induced toxicity.