Role Of DNA Methylation In Regulation Research Articles

Epigenetic regulation of organ-specific functions in Mikania micrantha and Mikania cordata: insights from DNA methylation and siRNA integration

BackgroundDNA methylation is a crucial epigenetic mechanism that regulates gene expression during plant growth and development. However, the role of DNA methylation in regulating the organ-specific functions of the invasive weed Mikania micrantha remains unknown.ResultsHere, we generated DNA methylation profiles for M. micrantha and a local congeneric species, Mikania cordata, in three vegetative organs (root, stem, and leaf) using whole-genome bisulfite sequencing. The results showed both differences and conservation in methylation levels and patterns between the two species. Combined with transcriptome data, we found that DNA methylation generally inhibited gene expression, with varying effects depending on the genomic region and sequence context (CG, CHG, and CHH). Genes overlapping with differentially methylated regions (DMRs) were more likely to be differentially expressed between organs, and DMR-associated upregulated differentially expressed genes (DEGs) were enriched in organ-specific pathways. A comparison between photosynthetic (leaf) and non-photosynthetic (root) organs of M. micrantha further confirmed the regulatory role of DNA methylation in leaf-specific photosynthesis. Integrating small RNA-Seq data revealed that 24-nt small interfering RNAs (siRNAs) were associated with CHH methylation in gene-rich regions and regulated CHH methylation in the flanking regions of photosynthesis-related genes.ConclusionThis study provides insights into the complex regulatory role of DNA methylation and siRNAs in organ-specific functions and offers valuable information for exploring the invasive characteristics of M. micrantha from an epigenetic perspective.

Methyl Donor Micronutrients Orchestrate Lipid Metabolism: The Role of DNA Methylation Modification

DNA methylation, a key epigenetic modification, plays a crucial role in regulating lipid metabolism. Consistent correlations have been observed between aberrant DNA methylation patterns and lipid metabolic disorders. Emerging evidence indicates that methyl donor micronutrients could influence DNA methylation patterns, consequently exerting an influence on lipid metabolism. Specifically, the deficiency or excesses of methyl donor micronutrients (folate, choline, betaine, B vitamins and methionine) have been associated with altered DNA methylation patterns linked to lipid metabolism. These alteration in DNA methylation levels, occurring globally and within promoter regions, could affect gene expression related to lipid metabolism. However, the mechanisms through which methyl donor micronutrients regulate lipid metabolism via the DNA methylation modification and the role of methyl donor micronutrients supplementation on DNA methylation profiles remain unclear. In this review, we summarized the regulatory role of DNA methylation in lipid metabolism, and highlighted recent findings investigating the impact of methyl donor micronutrients on lipid metabolism, as well as DNA methylation-mediated adipogenesis and adipose deposition. Taken together, this review deepened our understanding of how the complex interplay between methyl donor micronutrients, DNA methylation, and lipid metabolism, and provides valuable information for accurately regulating lipid metabolism of livestock and poultry, thereby improving meat quality, and promoting the development of animal husbandry.

Decoding the Root Lignification Mechanism of Angelica sinensis through Genome-wide Methylation Analysis.

Angelica sinensis is a traditional Chinese herbal medicine with significant economic and medicinal value. However, early bolting and flowering can occur during the second year of the vegetative growth period, rendering the roots unviable for medicinal use and resulting in substantial economic losses. Consequently, the growing interest in studying the molecular mechanisms underlying early bolting or increased root lignification in A. sinensis. Here, we conducted whole-genome bisulfite sequencing and observed an increase in whole-genome DNA methylation levels on chromosomes after bolting in A. sinensis. Comparative analysis revealed methylation patterns in the upstream, gene body, and downstream regions in the context of CG, CHG, and CHH, suggesting a possible association between CHH-type methylation of promoters and phenylpropanoid biosynthesis. Furthermore, joint analysis of transcriptomic and methylomics data revealed a positive correlation between DNA methylation and gene expression. We identified the hyperDMR gene in the CHH background within the promoter region; this gene is also a key gene (AsCOMT1), exhibiting dual catalytic activity and facilitating the synthesis of both ferulic acid and lignin. Enzyme kinetic analysis demonstrated that AsCOMT1 preferentially catalyzes the synthesis of lignin monomer precursors. These findings highlight the important regulatory role of DNA methylation in bolting and the synthesis of secondary metabolites in A. sinensis, providing valuable insights into the underlying molecular mechanisms. Therefore, as DNA methylation plays an important regulatory role in A. sinensis bolting and secondary metabolite synthesis, it has potential significance in the analysis of the underlying molecular mechanism.

Comprehensive Analysis of Placental DNA Methylation Changes and Fetal Birth Weight in Pigs.

Birth weight is a complex multifactorial trait relevant to health states and disease risks in later life. The placenta is essential for proper fetal growth and facilitates gas, nutrient, and waste exchange between the mother and developing fetus. How changes in placental DNA methylation affect fetal birth weight remains to be fully elucidated. In this study, we used whole-genome bisulfite sequencing and RNA sequencing to reveal a global map of DNA methylation and gene expression changes between the placentas of highest birth weight and lowest birth weight piglets in the same litters. The transcriptome analysis identified 1682 differential expressed genes and revealed key transcriptional properties in distinct placentas. We also identified key transcription factors that may drive the differences in DNA methylome patterns between placentas. The decrease in DNA methylation level in the promoter was associated with the transcriptional activation of genes associated with angiogenesis, extracellular matrix remodeling, and transmembrane transport. Our results revealed the regulatory role of DNA methylation in gene transcription activity leading to the differences in placental morphological structures and birth weights of piglets. These results could provide novel clues to clarify the underlying regulatory mechanisms of placental development and fetal growth.

Elucidating the Role of Transcriptomic Networks and DNA Methylation in Collagen Deposition of Dezhou Donkey Skin.

DNA methylation represents a predominant epigenetic modification with broad implications in various biological functions. Its role is particularly significant in the process of collagen deposition, a fundamental aspect of dermal development in donkeys. Despite its critical involvement, the mechanistic insights into how DNA methylation influences collagen deposition in donkey skin remain limited. In this study, we employed whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) to investigate the epigenetic landscape and gene expression profiles in the dorsal skin tissues of Dezhou donkeys across three developmental stages: embryonic (YD), juvenile (2-year-old, MD), and mature (8-year-old, OD). Our analysis identified numerous differentially methylated genes that play pivotal roles in skin collagen deposition and overall skin maturation, including but not limited to COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, GLUL, SFRP2, FOSL1, SERPINE1, MMP1, MMP2, MMP9, and MMP13. Notably, we observed an inverse relationship between gene expression and DNA methylation proximal to transcription start sites (TSSs), whereas a direct correlation was detected in regions close to transcription termination sites (TTSs). Detailed bisulfite sequencing analyses of the COL1A1 promoter region revealed a low methylation status during the embryonic stage, correlating with elevated transcriptional activity and gene expression levels. Collectively, our findings elucidate key genetic markers associated with collagen deposition in the skin of Dezhou donkeys, underscoring the significant regulatory role of DNA methylation. This research work contributes to the foundational knowledge necessary for the genetic improvement and selective breeding of Dezhou donkeys, aiming to enhance skin quality attributes.

Methylation-directed regulatory networks determine enhancing and silencing of mutation disease driver genes and explain inter-patient expression variation

BackgroundCommon diseases manifest differentially between patients, but the genetic origin of this variation remains unclear. To explore possible involvement of gene transcriptional-variation, we produce a DNA methylation-oriented, driver-gene-wide dataset of regulatory elements in human glioblastomas and study their effect on inter-patient gene expression variation.ResultsIn 175 of 177 analyzed gene regulatory domains, transcriptional enhancers and silencers are intermixed. Under experimental conditions, DNA methylation induces enhancers to alter their enhancing effects or convert into silencers, while silencers are affected inversely. High-resolution mapping of the association between DNA methylation and gene expression in intact genomes reveals methylation-related regulatory units (average size = 915.1 base-pairs). Upon increased methylation of these units, their target-genes either increased or decreased in expression. Gene-enhancing and silencing units constitute cis-regulatory networks of genes. Mathematical modeling of the networks highlights indicative methylation sites, which signified the effect of key regulatory units, and add up to make the overall transcriptional effect of the network. Methylation variation in these sites effectively describe inter-patient expression variation and, compared with DNA sequence-alterations, appears as a major contributor of gene-expression variation among glioblastoma patients.ConclusionsWe describe complex cis-regulatory networks, which determine gene expression by summing the effects of positive and negative transcriptional inputs. In these networks, DNA methylation induces both enhancing and silencing effects, depending on the context. The revealed mechanism sheds light on the regulatory role of DNA methylation, explains inter-individual gene-expression variation, and opens the way for monitoring the driving forces behind deferential courses of cancer and other diseases.

Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters.

In the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene. We constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region. The DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors. These findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

Pan-cancer analysis revealed H3K4me1 at bivalent promoters premarks DNA hypermethylation during tumor development and identified the regulatory role of DNA methylation in relation to histone modifications

BackgroundDNA hypermethylation at promoter CpG islands (CGIs) is a hallmark of cancers and could lead to dysregulation of gene expression in the development of cancers, however, its dynamics and regulatory mechanisms remain elusive. Bivalent genes, that direct development and differentiation of stem cells, are found to be frequent targets of hypermethylation in cancers.ResultsHere we performed comprehensive analysis across multiple cancer types and identified that the decrease in H3K4me1 levels coincides with DNA hypermethylation at the bivalent promoter CGIs during tumorigenesis. Removal of DNA hypermethylation leads to increment of H3K4me1 at promoter CGIs with preference for bivalent genes. Nevertheless, the alteration of H3K4me1 by overexpressing or knockout LSD1, the demethylase of H3K4, doesn’t change the level or pattern of DNA methylation. Moreover, LSD1 was found to regulate the expression of a bivalent gene OVOL2 to promote tumorigenesis. Knockdown of OVOL2 in LSD1 knockout HCT116 cells restored the cancer cell phenotype.ConclusionIn summary, our work identified a universal indicator that can pre-mark DNA hypermethylation in cancer cells, and dissected the interplay between H3K4me1 and DNA hypermethylation in detail. Current study also reveals a novel mechanism underlying the oncogenic role of LSD1, providing clues for cancer therapies.

LINE-1 ORF1p as a candidate biomarker in high grade serous ovarian carcinoma

Long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p) expression is a common feature of many cancer types, including high-grade serous ovarian carcinoma (HGSOC). Here, we report that ORF1p is not only expressed but also released by ovarian cancer and primary tumor cells. Immuno-multiple reaction monitoring-mass spectrometry assays showed that released ORF1p is confidently detectable in conditioned media, ascites, and patients’ plasma, implicating ORF1p as a potential biomarker. Interestingly, ORF1p expression is detectable in fallopian tube (FT) epithelial precursors of HGSOC but not in benign FT, suggesting that ORF1p expression in an early event in HGSOC development. Finally, treatment of FT cells with DNA methyltransferase inhibitors led to robust expression and release of ORF1p, validating the regulatory role of DNA methylation in LINE-1 repression in non-tumorigenic tissue.

DNA Methylation-Dependent Restriction of Tyrosine Hydroxylase Contributes to Pancreatic β-Cell Heterogeneity.

The molecular and functional heterogeneity of pancreatic β-cells is well recognized, but the underlying mechanisms remain unclear. Pancreatic islets harbor a subset of β-cells that co-express tyrosine hydroxylase (TH), an enzyme involved in synthesis of catecholamines that repress insulin secretion. Restriction of the TH+ β-cells within islets is essential for appropriate function in mice, such that a higher proportion of these cells corresponds to reduced insulin secretion. Here, we use these cells as a model to dissect the developmental control of β-cell heterogeneity. We define the specific molecular and metabolic characteristics of TH+ β-cells and show differences in their developmental restriction in mice and humans. We show that TH expression in β-cells is restricted by DNA methylation during β-cell differentiation. Ablation of de novo DNA methyltransferase Dnmt3a in the embryonic progenitors results in a dramatic increase in the proportion of TH+ β-cells, whereas β-cell-specific ablation of Dnmt3a does not. We demonstrate that maintenance of Th promoter methylation is essential for its continued restriction in postnatal β-cells. Loss of Th promoter methylation in response to chronic overnutrition increases the number of TH+ β-cells, corresponding to impaired β-cell function. These results reveal a regulatory role of DNA methylation in determining β-cell heterogeneity.

Myeloid-derived suppressor cell: A crucial player in autoimmune diseases.

Myeloid-derived suppressor cells (MDSCs) are identified as a highly heterogeneous group of immature cells derived from bone marrow and play critical immunosuppressive functions in autoimmune diseases. Accumulating evidence indicates that the pathophysiology of autoimmune diseases was closely related to genetic mutations and epigenetic modifications, with the latter more common. Epigenetic modifications, which involve DNA methylation, covalent histone modification, and non-coding RNA-mediated regulation, refer to inheritable and potentially reversible changes in DNA and chromatin that regulate gene expression without altering the DNA sequence. Recently, numerous reports have shown that epigenetic modifications in MDSCs play important roles in the differentiation and development of MDSCs and their suppressive functions. The molecular mechanisms of differentiation and development of MDSCs and their regulatory roles in the initiation and progression of autoimmune diseases have been extensively studied, but the exact function of MDSCs remains controversial. Therefore, the biological and epigenetic regulation of MDSCs in autoimmune diseases still needs to be further characterized. This review provides a detailed summary of the current research on the regulatory roles of DNA methylation, histone modifications, and non-coding RNAs in the development and immunosuppressive activity of MDSCs, and further summarizes the distinct role of MDSCs in the pathogenesis of autoimmune diseases, in order to provide help for the diagnosis and treatment of diseases from the perspective of epigenetic regulation of MDSCs.

CHH methylation of genes associated with fatty acid and jasmonate biosynthesis contributes to cold tolerance in autotetraploids of Poncirus trifoliata.

Polyploids have elevated stress tolerance, but the underlying mechanisms remain largely elusive. In this study, we showed that naturally occurring tetraploid plants of trifoliate orange (Poncirus trifoliata (L.) Raf.) exhibited enhanced cold tolerance relative to their diploid progenitors. Transcriptome analysis revealed that whole-genome duplication was associated with higher expression levels of a range of well-characterized cold stress-responsive genes. Global DNA methylation profiling demonstrated that the tetraploids underwent more extensive DNA demethylation in comparison with the diploids under cold stress. CHH methylation in the promoters was associated with up-regulation of related genes, whereas CG, CHG, and CHH methylation in the 3'-regions was relevant to gene down-regulation. Of note, genes involved in unsaturated fatty acids (UFAs) and jasmonate (JA) biosynthesis in the tetraploids displayed different CHH methylation in the gene flanking regions and were prominently up-regulated, consistent with greater accumulation of UFAs and JA when exposed to the cold stress. Collectively, our findings explored the difference in cold stress response between diploids and tetraploids at both transcriptional and epigenetic levels, and gained new insight into the molecular mechanisms underlying enhanced cold tolerance of the tetraploid. These results contribute to uncovering a novel regulatory role of DNA methylation in better cold tolerance of polyploids.

DNA methylation of the MAP3K10 gene may participate in the development of intracranial aneurysm

BackgroundThe goal of this study was to explore the association between mitogen-activated protein kinase kinase kinase 10 (MAP3K10) methylation and blood lipid levels and intracranial aneurysm (IA) risk. Materials and methodsA total of 96 age- and sex-matched investigators participated in the assessment of MAP3K10 methylation. Fourteen CpG sites of the MAP3K10 gene were selected for methylated-pyrosequencing. Human brain vascular smooth muscle cell was used to assess the regulatory role of DNA methylation in MAP3K10 gene transcription. ResultsMAP3K10 mean methylation was positively correlated with triglyceride (TG, r = 0.388; p = 0.007) in men, but negatively correlated with TG (r = -0.434; p = 0.002) in women. MAP3K10 methylation in patients with IA was significantly lower than that in controls (p < 0.05), and this phenomenon was more significant in women (12 CpG sites presented significance at p < 0.05). MAP3K10 methylation might be a potential predictor of the risk of IA (CpG1, AUC = 0.81, p < 0.001; mean methylation, AUC = 0.69, p = 0.001). The predictive value was also more significant in women (CpG1: AUC = 0.86, p < 0.001; mean methylation: AUC = 0.73, p = 0.006). No significant association was found between DNA methylation and MAP3K10 gene transcription in vitro experiment. ConclusionPatients with IA had lower MAP3K10 methylation levels than healthy controls. MAP3K10 methylation may be a potential predictor of IA risk, particularly in women.

PSXIV-9 Identification of Differential Methylation Regions as Candidate Discriminant Markers of Bovine Subclinical Mastitis Caused by Staphylococcus Aureus

Abstract Staphylococcus aureus (S. aureus) is one of the most prevalent pathogens of bovine subclinical mastitis and a challenging pathogen in dairy production. DNA methylation involvement in regulating mammary gland inflammatory defense has been documented. This study adopted whole genome bisulfite sequencing and RNA-sequencing technologies to profile the genome-wide DNA methylation and transcriptome landscapes, respectively, of milk somatic cells (MSCs) from 15 cows with S. aureus-induced subclinical mastitis (SACs) and 13 healthy cows (HCs). Bioinformatics processing of data with standard tools indicated that the global DNA methylation patterns of MSCs demonstrated inverse correlations between gene expression and DNA methylation levels at promoter, first exon and first intron regions, suggesting potential regulatory roles of DNA methylation in transcriptional activities. Totally, 26,289 differential methylation regions (DMRs) were identified between SACs and HCs (1,000 bp window, 1,000 bp step, q-value &amp;lt; 0.05, &amp;gt;20% methylation difference and ≥3 differentially methylated cytosines). A total of 887, 334 and 3,414 DMRs were overlapped with promoter, first exon, and first intron regions, respectively, including 363 DMRs harboring transcription start sites. Among them, the methylation levels of 956 DMRs were strongly correlated with the expression levels of their overlapped genes (|Spearman’s correlation coefficient| &amp;gt;0.5, adjusted-p-value&amp;lt; 0.05). Functional annotation of the overlapped genes indicated significant (adjusted-p-value &amp;lt; 0.05) enrichment in 18 GO terms and 1 KEGG pathway related to cellular activities, notably in cell migration, epithelial cell differentiation and tight junction, among others (Figure 1). These results suggest the involvement of DNA methylation in mammary gland defense against S. aureus-invasion. Applying DIABLO method to integrate correlated DMRs and genes, a total of 20 DMR and 10 gene candidate markers were found to discriminate SACs from HCs. The identified candidate markers could serve as reference for enhancing breeding for mastitis resistance and for developing new mastitis management strategies.

Differential DNA methylation in Pacific oyster reproductive tissue in response to ocean acidification

BackgroundThere is a need to investigate mechanisms of phenotypic plasticity in marine invertebrates as negative effects of climate change, like ocean acidification, are experienced by coastal ecosystems. Environmentally-induced changes to the methylome may regulate gene expression, but methylome responses can be species- and tissue-specific. Tissue-specificity has implications for gonad tissue, as gonad-specific methylation patterns may be inherited by offspring. We used the Pacific oyster (Crassostrea gigas) — a model for understanding pH impacts on bivalve molecular physiology due to its genomic resources and importance in global aquaculture— to assess how low pH could impact the gonad methylome. Oysters were exposed to either low pH (7.31 ± 0.02) or ambient pH (7.82 ± 0.02) conditions for 7 weeks. Whole genome bisulfite sequencing was used to identify methylated regions in female oyster gonad samples. C- > T single nucleotide polymorphisms were identified and removed to ensure accurate methylation characterization.ResultsAnalysis of gonad methylomes revealed a total of 1284 differentially methylated loci (DML) found primarily in genes, with several genes containing multiple DML. Gene ontologies for genes containing DML were involved in development and stress response, suggesting methylation may promote gonad growth homeostasis in low pH conditions. Additionally, several of these genes were associated with cytoskeletal structure regulation, metabolism, and protein ubiquitination — commonly-observed responses to ocean acidification. Comparison of these DML with other Crassostrea spp. exposed to ocean acidification demonstrates that similar pathways, but not identical genes, are impacted by methylation.ConclusionsOur work suggests DNA methylation may have a regulatory role in gonad and larval development, which would shape adult and offspring responses to low pH stress. Combined with existing molluscan methylome research, our work further supports the need for tissue- and species-specific studies to understand the potential regulatory role of DNA methylation.

Complex regulatory role of DNA methylation in caste- and age-specific expression of a termite.

The reproductive castes of eusocial insects are often characterized by extreme lifespans and reproductive output, indicating an absence of the fecundity/longevity trade-off. The role of DNA methylation in the regulation of caste- and age-specific gene expression in eusocial insects is controversial. While some studies find a clear link to caste formation in honeybees and ants, others find no correlation when replication is increased across independent colonies. Although recent studies have identified transcription patterns involved in the maintenance of high reproduction throughout the long lives of queens, the role of DNA methylation in the regulation of these genes is unknown. We carried out a comparative analysis of DNA methylation in the regulation of caste-specific transcription and its importance for the regulation of fertility and longevity in queens of the higher termite Macrotermes natalensis. We found evidence for significant, well-regulated changes in DNA methylation in mature compared to young queens, especially in several genes related to ageing and fecundity in mature queens. We also found a strong link between methylation and caste-specific alternative splicing. This study reveals a complex regulatory role of fat body DNA methylation both in the division of labour in termites, and during the reproductive maturation of queens.

Regulatory Role of DNA Methylation and Its Significance in Plants

DNA methylation is a well-known epigenetic modification that is essential for gene regulation and genome stability. Anomalies in plant development can result from aberrant DNA methylation patterns. DNA methylation is much more important in plants with more complicated genomes when it comes to growth and abiotic stress tolerance. Dynamic regulation via de novo methylation, maintenance of methylation, and active demethylation, which are catalysed by diverse enzymes that are targeted by different regulatory mechanisms, results in a unique DNA methylation state. We explain DNA methylation in plants, including methylating and demethylating enzymes and regulatory changes, as well as the coordination of methylation and demethylation activities by a mechanism known as the methylstat. We also explain the roles of DNA methylation in regulating transposon silencing, gene expression, and chromosome interactions, as well as the intervention of DNA methylation in plant responses to biotic and abiotic stresses.

Whole-Genome DNA Methylation Analysis in Brassica rapa subsp. perviridis in Response to Albugo candida Infection

DNA methylation is an epigenetic mark associated with several mechanisms in plants including immunity mechanisms. However, little is known about the regulatory role of DNA methylation in the resistance response of Brassica species against fungal diseases. White rust, caused by the fungus Albugo candida, is one of the most widespread and destructive diseases of all the cultivated Brassica species, particularly Brassica rapa L. and Brassica juncea (L.) Czern and Coss. Here, we investigate whole-genome DNA methylation modifications of B. rapa subsp. perviridis in response to white rust. As a result, 233 and 275 differentially methylated regions (DMRs) in the susceptible cultivar “Misugi” and the resistant cultivar “Nanane” were identified, respectively. In both cultivars, more than half of the DMRs were associated with genes (DMR-genes). Gene expression analysis showed that 13 of these genes were also differentially expressed between control and infected samples. Gene ontology enrichment analysis of DMR genes revealed their involvement in various biological processes including defense mechanisms. DMRs were unevenly distributed around genes in susceptible and resistant cultivars. In “Misugi,” DMRs tended to be located within genes, while in “Nanane,” DMRs tended to be located up and downstream of the genes. However, CG DMRs were predominantly located within genes in both cultivars. Transposable elements also showed association with all three sequence contexts of DMRs but predominantly with CHG and CHH DMRs in both cultivars. Our findings indicate the occurrence of DNA methylation modifications in B. rapa in response to white rust infection and suggest a potential regulatory role of DNA methylation modification in defense mechanisms which could be exploited to improve disease resistance.

Global DNA Methylation and mRNA-miRNA Variations Activated by Heat Shock Boost Early Microspore Embryogenesis in Cabbage (Brassica oleracea).

Microspore culture, a type of haploid breeding, is extensively used in the cultivation of cruciferous crops such as cabbage. Heat shock (HS) treatment is essential to improve the embryo rate during the culture process; however, its molecular role in boosting early microspore embryogenesis (ME) remains unknown. Here we combined DNA methylation levels, miRNAs, and transcriptome profiles in isolated microspores of cabbage ‘01-88’ under HS (32 °C for 24 h) and normal temperature (25 °C for 24 h) to investigate the regulatory roles of DNA methylation and miRNA in early ME. Global methylation levels were significantly different in the two pre-treatments, and 508 differentially methylated regions (DMRs) were identified; 59.92% of DMRs were correlated with transcripts, and 39.43% of miRNA locus were associated with methylation levels. Significantly, the association analysis revealed that 31 differentially expressed genes (DEGs) were targeted by methylation and miRNA and were mainly involved in the reactive oxygen species (ROS) response and abscisic acid (ABA) signaling, indicating that HS induced DNA methylation, and miRNA might affect ME by influencing ROS and ABA. This study revealed that DNA methylation and miRNA interfered with ME by modulating key genes and pathways, which could broaden our understanding of the molecular regulation of ME induced by HS pre-treatment.

Transcriptome Sequencing Highlights the Regulatory Role of DNA Methylation in Immune-Related Genes' Expression of Chinese Oak Silkworm, Antheraea pernyi.

Simple SummaryAntheraea pernyi is an important lepidopteran that has been used as a model insect for various physiological systems. DNA methylation has recently been discovered to control a variety of physiological processes in animals throughout their lives. We investigated the effect of DNA methylation on the innate immune system of A. pernyi and found that DNA methylation inhibition modifies gene expression, especially in immune-related genes. Finally, our data suggest that DNA methylation may regulate gene expression in insects, and microbial infection likely induces DNA methylation of the host genome.Antheraea pernyi is an important lepidopteran used as a model insect species to investigate immune responses, development, and metabolism modulation. DNA methylation has recently been found to control various physiological processes throughout the life of animals; however, DNA methylation and its effect on the physiology of insects have been poorly investigated so far. In the present study, to better understand DNA methylation and its biological role in the immune system, we analyzed transcriptome profiles of A. pernyi pupae following DNA methylation inhibitor injection and Gram-positive bacteria stimulation. We then compared the profiles with a control group. We identified a total of 55,131 unigenes from the RNA sequence data. A comparison of unigene expression profiles showed that a total of 680 were up-regulated and 631 unigenes were down-regulated in the DNA-methylation-inhibition-bacteria-infected group compared to the control group (only bacteria-injected pupae), respectively. Here, we focused on the immune-related differentially expressed genes (DEGs) and screened 10 genes that contribute to immune responses with an up-regulation trend, suggesting that microbial pathogens evade host immunity by increasing DNA methylation of the host genome. Furthermore, several other unigenes related to other pathways were also changed, as shown in the KEGG analysis. Taken together, our data revealed that DNA methylation seems to play a crucial biological role in the regulation of gene expression in insects, and that infection may enhance the host genome DNA methylation by a yet-unknown mechanism.