Genome-wide analysis of DNA methylation in response to light and salt stress in a halophyte with single cell C4 pathway.

DNA methylation is an important epigenetic modification that has been shown to be associated with responses to non-biological stressors. However, there is currently no research on DNA methylation in response to environmental signals in shrimp. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles and differentially expressed genes between two strains of Litopenaeus vannamei with significantly different cold tolerance through whole genome bisulfite sequencing (WGBS) and transcriptome sequencing. Between Lv-C and Lv-T (constant temperature of 28 °C and low temperatures of 18 °C and 10 °C) under cytosine-guanine (CG) environments, 39,100 differentially methylated regions (DMRs) were identified, corresponding to 9302 DMR-related genes (DMRGs). The DMRs were mainly located in the gene body (exons and introns). Gene Ontology (GO) analysis showed that these DMRGs were significantly enriched in cell parts, catalytic activity, and metabolic processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed significant enrichment of these DMRGs in pathways such as proteasome (ko03050), oxidative phosphorylation (ko00190), mTOR signaling pathway (ko04150), fatty acid metabolism (ko01212), and fatty acid degradation (ko00071). The comprehensive results suggested that L. vannamei mainly regulates gene expression in response to low temperatures through hypermethylation or demethylation of some genes involved in thermogenesis, glycolysis, the autophagy pathway, the peroxisome, and drug metabolism pathways. These results provide important clues for studying DNA methylation patterns and identifying cold tolerance genes in shrimp.

As a salt-adaptive plant, the date palm (Phoenix dactylifera L.) requires a suitable mechanism to adapt to the stress of saline soils. There is growing evidence that DNA methylation plays an important role in regulating gene expression in response to abiotic stresses, including salinity. Thus, the present study sought to examine the differential methylation status that occurs in the date palm genome when plants are exposed to salinity, and to identify salinity responsive genes that are regulated by DNA methylation. To achieve these, whole-genome bisulfite sequencing (WGBS) was employed and mRNA was sequenced from salinity-treated and untreated roots. The WGBS analysis included 324,987,795 and 317,056,091 total reads of the control and the salinity-treated samples, respectively. The analysis covered about 81% of the total genomic DNA with about 40% of mapping efficiency of the sequenced reads and an average read depth of 17-fold coverage per DNA strand, and with a bisulfite conversion rate of around 99%. The level of methylation within the differentially methylated regions (DMRs) was significantly (p < 0.05, FDR ≤ 0.05) increased in response to salinity specifically at the mCHG and mCHH sequence contexts. Consistently, the mass spectrometry and the enzyme-linked immunosorbent assay (ELISA) showed that there was a significant (p < 0.05) increase in the global DNA methylation in response to salinity. mRNA sequencing revealed the presence of 6,405 differentially regulated genes with a significant value (p < 0.001, FDR ≤ 0.05) in response to salinity. Integration of high-resolution methylome and transcriptome analyses revealed a negative correlation between mCG methylation located within the promoters and the gene expression, while a positive correlation was noticed between mCHG/mCHH methylation rations and gene expression specifically when plants grew under control conditions. Therefore, the methylome and transcriptome relationships vary based on the methylated sequence context, the methylated region within the gene, the protein-coding ability of the gene, and the salinity treatment. These results provide insights into interplay among DNA methylation and gene expression, and highlight the effect of salinity on the nature of this relationship, which may involve other genetic and epigenetic players under salt stress conditions. The results obtained from this project provide the first draft map of the differential methylome and transcriptome of date palm when exposed to an abiotic stress.

Previous evidence demonstrated DNA methylation changes in response to stress in plants, showing rapid changes within a limited time frame. Exposure to self-DNA inhibits seedling root elongation, and it was shown that it causes changes in CG DNA methylation in Lactuca sativa. We assessed cytosine methylation changes and associated gene expression patterns in roots of Arabidopsis thaliana Col-0 seedlings exposed to self-DNA for 6 and 24 h. We used whole genome bisulfite sequencing (WGBS) and RNA-seq analyses to assess genomic cytosine methylation and corresponding gene expression, respectively, on DNA and RNA extracted with commercial kits from roots exposed to self-DNA by an original setup. Fifteen hundred roots replicates, including the control in distilled water, were collected after exposure. Sequencing was performed on a NovaSeq 6000 platform and Ultralow Methyl-Seq System for RNA and DNA WGBS, respectively. Gene expression in roots exposed to self-DNA differed from that of untreated controls, with a total of 305 genes differentially expressed and 87 ontologies enriched in at least one treatment vs. control comparison, and particularly after 24 h of exposure. DNA methylation, particularly in CHG and CHH contexts, was also different, with hyper- and hypomethylation prevailing in treatments vs. controls at 6 h and 24 h, respectively. Differentially expressed genes (DEGs) analysis, Gene Ontology (GO) enrichment analysis, and differentially methylated regions (DMRs) analysis, provided an integrated understanding of the changes associated with self-DNA exposure. Our results suggest differential gene expression associated with DNA methylation in response to self-DNA exposure in A. thaliana roots, enhanced after prolonged exposure. Main functional indications of association between DNA methylation and gene expression involved hypomethylation and downregulation of genes related to nucleotide/nucleoside metabolism (ATP synthase subunit) and cell wall structure (XyG synthase), consistent with previous observations from metabolomics and physiological studies. Further confirmation of these findings will contribute to improving our understanding of the plant molecular response to self-DNA and its implications in stress responses.

Emerging evidence published over the past decade has highlighted the role of DNA methylation in skeletal muscle function and health, including as an epigenetic transducer of the adaptive response to exercise. In this review, we aim to synthesize the latest findings in this field to highlight: (1) the shifting understanding of the genomic localization of altered DNA methylation in response to acute and chronic aerobic and resistance exercise in skeletal muscle (e.g., promoter, gene bodies, enhancers, intergenic regions, un-annotated regions, and genome-wide methylation); (2) how these global/regional methylation changes relate to transcriptional activity following exercise; and (3) the factors (e.g., individual demographic or genetic features, dietary, training history, exercise parameters, local epigenetic characteristics, circulating hormones) demonstrated to alter both the pattern of DNA methylation after exercise, and the relationship between DNA methylation and gene expression. Finally, we discuss the changes in non-CpG methylation and 5-hydroxymethylation after exercise, as well as the importance of emerging single-cell analyses to future studies-areas of increasing focus in the field of epigenetics. We anticipate that this review will help generate a framework for clinicians and researchers to begin developing and testing exercise interventions designed to generate targeted changes in DNA methylation as part of a personalized exercise regimen.

Whole-genome DNA methylation profiling revealed epigenetic regulation of NF-κB signaling pathway involved in response to Vibrio alginolyticus infection in the Pacific oyster, Crassostrea gigas

BackgroundDNA methylation is an important epigenetic modification. It can regulate the expression of many key genes without changing the primary structure of the genomic DNA, and plays a vital role in the growth and development of the organism. The genome-wide DNA methylation profile of the cytoplasmic male sterile (CMS) line in soybean has not been reported so far.ResultsIn this study, genome-wide comparative analysis of DNA methylation between soybean CMS line NJCMS5A and its maintainer NJCMS5B was conducted by whole-genome bisulfite sequencing. The results showed 3527 differentially methylated regions (DMRs) and 485 differentially methylated genes (DMGs), including 353 high-credible methylated genes, 56 methylated genes coding unknown protein and 76 novel methylated genes with no known function were identified. Among them, 25 DMRs were further validated that the genome-wide DNA methylation data were reliable through bisulfite treatment, and 9 DMRs were confirmed the relationship between DNA methylation and gene expression by qRT-PCR. Finally, 8 key DMGs possibly associated with soybean CMS were identified.ConclusionsGenome-wide DNA methylation profile of the soybean CMS line NJCMS5A and its maintainer NJCMS5B was obtained for the first time. Several specific DMGs which participated in pollen and flower development were further identified to be probably associated with soybean CMS. This study will contribute to further understanding of the molecular mechanism behind soybean CMS.

Cytokinins play important roles in the growth and development of plants. Physiological and photosynthetic characteristics are common indicators to measure the growth and development in plants. However, few reports have described the molecular mechanisms of physiological and photosynthetic changes in response to cytokinin, particularly in woody plants. DNA methylation is an essential epigenetic modification that dynamically regulates gene expression in response to the external environment. In this study, we examined genome-wide DNA methylation variation and transcriptional variation in poplar (Populus tomentosa) after short-term treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA). We identified 460 significantly differentially methylated regions (DMRs) in response to 6-BA treatment. Transcriptome analysis showed that 339 protein-coding genes, 262 long non-coding RNAs (lncRNAs), and 15,793 24-nt small interfering RNAs (siRNAs) were differentially expressed under 6-BA treatment. Among these, 79% were differentially expressed between alleles in P. tomentosa, and 102,819 allele-specific expression (ASE) loci in 19,200 genes were detected showing differences in ASE levels after 6-BA treatment. Combined DNA methylation and gene expression analysis demonstrated that DNA methylation plays an important role in regulating allele-specific gene expression. To further investigate the relationship between these 6-BA-responsive genes and phenotypic variation, we performed SNP analysis of 460 6-BA-responsive DMRs via re-sequencing using a natural population of P. tomentosa, and we identified 206 SNPs that were significantly associated with growth and wood properties. Association analysis indicated that 53% of loci with allele-specific expression had primarily dominant effects on poplar traits. Our comprehensive analyses of P. tomentosa DNA methylation and the regulation of allele-specific gene expression suggest that DNA methylation is an important regulator of imbalanced expression between allelic loci.

DNA methylation has been referred as an important player in plant genomic responses to environmental stresses but correlations between the methylome plasticity and specific traits of interest are still far from being understood. In this study, we inspected global DNA methylation levels in salt tolerant and sensitive rice varieties upon salt stress imposition. Global DNA methylation was quantified using the 5-methylcytosine (5mC) antibody and an ELISA-based technique, which is an affordable and quite pioneer assay in plants, and in situ imaging of methylation sites in interphase nuclei of tissue sections. Variations of global DNA methylation levels in response to salt stress were tissue- and genotype-dependent. We show a connection between a higher ability of DNA methylation adjustment levels and salt stress tolerance. The salt-tolerant rice variety Pokkali was remarkable in its ability to quickly relax DNA methylation in response to salt stress. In spite of the same tendency for reduction of global methylation under salinity, in the salt-sensitive rice variety IR29 such reduction was not statistically supported. In ‘Pokkali’, the salt stress-induced demethylation may be linked to active demethylation due to increased expression of DNA demethylases under salt stress. In ‘IR29’, the induction of both DNA demethylases and methyltransferases may explain the lower plasticity of DNA methylation. We further show that mutations for epigenetic regulators affected specific phenotypic parameters related to salinity tolerance, such as the root length and biomass. This work emphasizes the role of differential methylome flexibility between salt tolerant and salt sensitive rice varieties as an important player in salt stress tolerance, reinforcing the need to better understand the connection between epigenetic networks and plant responses to environmental stresses.

Study question Do DNA methylation changes occur in testicular germ cells (TGCs) from patients with impaired spermatogenesis? Summary answer TGCs from men with cryptozoospermia exhibit altered DNA methylation levels at several genomic regions, many of which are associated with genes involved in spermatogenesis. What is known already In the last 15 years, several studies have described DNA methylation changes in sperm of infertile men. More recently, using whole genome bisulfite sequencing (WGBS) we were able to refute these findings by demonstrating that somatic DNA contamination and genetic variation confound methylation studies in swim-up purified sperm of severely oligozoospermic men. However, it remains unknown whether altered DNA methylation plays a role during the development of the germ cells in the testes of these patients. Study design, size, duration For identifying DNA methylation differences associated with impaired spermatogenesis, we compared the TGC methylomes of men with cryptozoospermia (CZ) and men with obstructive azoospermia (n = 4 each), who had normal spermatogenesis and served as controls (CTR). Study participants were selected among an age-matched cohort of 24 CTR and 10 CZ. The selection was based on similar composition of the TGC suspension evaluated by ploidy analysis and absence of somatic DNA. Participants/materials, setting, methods TGCs were isolated from biopsies after short-term cell culture. Presence of somatic DNA was evaluated by analyzing the DNA methylation levels of H19, MEST, DDX4 and XIST. WGBS was performed at ∼14× coverage. Bioinformatic tools were used to compare global DNA methylation levels, identify differentially methylated regions (DMRs) and functionally annotate the DMRs. Single-cell RNA sequencing (scRNA-seq) was used to associate the DNA methylation changes to gene expression. Main results and the role of chance We could not identify any difference in the global DNA methylation level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 DMRs, 238 of which were hypermethylated in CZ (binominal test, p &amp;lt; 2.2 × 10–16). The DMRs are associated with 132 genes, 61 of which are known to be differentially expressed at various stages of spermatogenesis according to scRNA-seq studies. Almost all of the DMRs associated with the 61 genes are hypermethylated in CZ (63/67, p = 1.107 × 10–14). As assessed by scRNA-seq, 13 DMR-associated genes, which were mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter was hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, most of which downregulated in CZ, germ cell-specific enhancers may be affected. Limitations, reasons for caution The small sample size constitutes a limitation of this study. Furthermore, even though the cellular composition of samples was similar by ploidy analysis, we cannot rule out that the observed DNA methylation changes might be due to differences in the relative proportion of different germ cell types. Wider implications of the findings: Impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome, which points to an important role of DNA methylation in normal spermatogenesis. The DNA methylation changes may contribute to premature abortion of spermatogenesis and therefore not appear in mature sperm. Trial registration number N/A

Integrative analysis of whole genome bisulfite and transcriptome sequencing reveals the effect of sodium butyrate on DNA methylation in the differentiation of bovine skeletal muscle satellite cells

BackgroundInducing embryogenic callus with regenerative potential is a pivotal step in barley transformation. Our previous research suggests that epigenetic regulatory factors might influence barley callus formation and regeneration capacity, though the exact mechanisms remain unclear.ResultsIn this study, we utilized RNA sequencing (RNA-seq) and whole-genome bisulfite sequencing (WGBS) to examine transcriptional and DNA methylome alterations during callus induction from immature embryos of the barley cultivar Golden Promise. Our findings revealed a slight decline in overall DNA methylation content and distinct 5-methylcytosine (5mC) enrichment patterns in CG, CHG, and CHH sequence contexts within genes and transposable elements. By integrating DNA methylation and transcriptome data, we identified differentially expressed genes (DEGs) associated with differentially methylated regions (DMRs) in the CG (879 DEGs), CHG (229 DEGs), and CHH (2020 DEGs) contexts. Notably, DMRs linked to 210, 94, and 1,214 DEGs were located in the 2 kb upstream regions in the CG, CHG, and CHH contexts, respectively. A negative correlation was observed between promoter methylation levels and transcript abundances of key regeneration-associated genes, such as HvKRP4, HvCYCD1;1, HvSCR, HvRAP2.6L/ERF113, HvWIND4, HvWOX5, HvE2Fa, HvPHV, and HvLBD16. This indicates a regulatory function of DNA methylation in transcriptional regulation during callus induction. Furthermore, treatment with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) suppressed callus formation. Comparative RNA sequencing analysis between control and treated groups revealed 2,628 and 1,224 DEGs potentially regulated by DNA methylation, at 2- and 9-days post-induction, respectively. These genes were primarily associated with cell cycle and abscisic acid signalling pathways, influenced directly and indirectly by the global reduction in DNA methylation induced by 5-Aza-dC treatment.ConclusionsThis study provides insights into the intricate relationship between DNA methylation and gene expression during barley callus formation. It could inform future efforts to enhance regeneration and transformation in this significant crop species.Clinical trial numberNot applicable.

Summary DNA methylation is dynamically involved in plant immunity, but little information is known about its roles in plant interactions with biotrophic fungi, especially in temperate grasses such as wheat (Triticum aestivum).Using wheat diploid progenitor Aegilops tauschii accession AL8/78, the genome of which has been sequenced, we assessed the extent of DNA methylation in response to infection with Blumeria graminis f. sp. tritici (Bgt), which causes powdery mildew.Upon Bgt infection, ARGONAUTE4a (AGO4a) was significantly downregulated in A. tauschii, which was accompanied by a substantial reduction in AGO4a‐sorted 24‐nt siRNA levels, especially for genes near transposable elements (TAGs). Bisulfite sequencing revealed abundant differentially methylated regions (DMRs) with CHH hypomethylation. TAGs bearing CHH‐hypomethylated DMRs were enriched for ‘response to stress’ functions, including receptor kinase, peroxidase, and pathogenesis‐related genes. Virus‐induced gene silencing (VIGS) of a DOMAINS REARRANGED METHYLASE 2 (DRM2) homolog enhanced plant resistance to Bgt. The effect of CHH hypomethylation was exemplified by the upregulation of a pathogenesis‐related β‐1,3‐glucanse gene implicated in Bgt defense.These findings support the idea that dynamic DNA methylation represents a regulatory layer in the complex mechanism of plant immunity, which could be exploited to improve disease resistance in common wheat.

Analysis of sunflower DNA methylation in response to salt and alkali stresses based on methylation-sensitive amplified polymorphisms

DNA methylation is a well-known epigenetic modification that plays a crucial role in gene regulation, but genome-wide analysis of DNA methylation remains technically challenging and costly. DNA methylation-dependent restriction enzymes can be used to restrict CpG methylation analysis to methylated regions of the genome only, which significantly reduces the required sequencing depth and simplifies subsequent bioinformatics analysis. Unfortunately, this approach has been hampered by complete digestion of DNA in CpG methylation-dense regions, resulting in fragments that are too small for accurate mapping. Here, we show that the activity of DNA methylation-dependent enzyme, LpnPI, is blocked by a fragment size smaller than 32 bp. This unique property prevents complete digestion of methylation-dense DNA and allows accurate genome-wide analysis of CpG methylation at single-nucleotide resolution. Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments revealed highly reproducible genome-wide CpG methylation profiles for >50% of all potentially methylated CpGs, at a sequencing depth less than one-tenth required for whole-genome bisulfite sequencing (WGBS). MeD-seq identified a high number of patient and tissue-specific differential methylated regions (DMRs) and revealed that patient-specific DMRs observed in both blood and buccal samples predict DNA methylation in other tissues and organs. We also observed highly variable DNA methylation at gene promoters on the inactive X Chromosome, indicating tissue-specific and interpatient-specific escape of X Chromosome inactivation. These findings highlight the potential of MeD-seq for high-throughput epigenetic profiling.

Genome-wide analysis of DNA methylation reveals significant impact of two typical high temperature stress on biological functions in Apostichopus japonicus