Gene Body Methylation Research Articles

Symbiont shuffling induces differential DNA methylation responses to thermal stress in the coral Montastraea cavernosa.

Algal symbiont shuffling in favour of more thermotolerant species has been shown to enhance coral resistance to heat-stress. Yet, the mechanistic underpinnings and long-term implications of these changes are poorly understood. This work studied the modifications in coral DNA methylation, an epigenetic mechanism involved in coral acclimatization, in response to symbiont manipulation and subsequent heat stress exposure. Symbiont composition was manipulated in the great star coral Montastraea cavernosa through controlled thermal bleaching and recovery, producing paired ramets of three genets dominated by either their native symbionts (genus Cladocopium) or the thermotolerant species (Durusdinium trenchi). Single-base genome-wide analyses showed significant modifications in DNA methylation concentrated in intergenic regions, introns and transposable elements. Remarkably, DNA methylation changes in response to heat stress were dependent on the dominant symbiont, with twice as many differentially methylated regions found in heat-stressed corals hosting different symbionts (Cladocopium vs. D. trenchii) compared to all other comparisons. Interestingly, while differential gene body methylation was not correlated with gene expression, an enrichment in differentially methylated regions was evident in repetitive genome regions. Overall, these results suggest that changes in algal symbionts favouring heat tolerant associations are accompanied by changes in DNA methylation in the coral host. The implications of these results for coral adaptation, along with future avenues of research based on current knowledge gaps, are discussed in the present work.

Common mechanisms cannot explain time- and dose-dependent DNA methylation changes in earthworms exposed to cadmium

DNA hypermethylation caused by environmental pollutants like cadmium (Cd) has already been demonstrated in many invertebrates, including earthworms. However, the exact epigenetic mechanisms that drive this hypermethylation are largely unknown and even basic DNA methylation and demethylation processes are hardly characterized. Therefore, we used an important bioindicator, the earthworm Lumbricus terrestris, as a model organism to determine time- and dose-dependent effects of Cd on global and gene-specific DNA methylation and its underlying mechanisms. We revealed Cd-induced adenine and cytosine hypermethylation using specific antibodies in dot blots and found that the methylation level of adenine compared to cytosine changed even to a bigger extent. However, the levels of hydroxymethylated cytosine did not differ between treatment groups. General methylation and demethylation components like methyltransferases (DNMT1 and 3), and ten-eleven translocation (TET) genes were confirmed in L. terrestris by quantitative RealTime PCR. However, neither gene expression, nor DNMT and TET enzyme activity showed significant differences in the Cd exposure groups. Using bisulfite conversion and sequencing, gene body methylation (gbm) of metallothionein 2 (MT2), one of the most important detoxification proteins, was characterized. Cd-dependent changes in MT2 gbm could, however, not be correlated to MT2 gene activity evaluated by quantitative RealTime PCR.Future directions as well as missing links are discussed in the present study hinting towards the importance of studying epigenetic marks and mechanistic insights in a broad variety of species to deepen our knowledge on the effects of changing environmental conditions.

Pleiotropic Effects of Functional MUC1 Variants on Cardiometabolic, Renal, and Hematological Traits in the Taiwanese Population

MUC1 is a transmembrane mucin involved in carcinogenesis and cell signaling. Functional MUC1 variants are associated with multiple metabolic and biochemical traits. This study investigated the association of functional MUC1 variants with MUC1 DNA methylation and various metabolic, biochemical, and hematological parameters. In total, 80,728 participants from the Taiwan Biobank were enrolled for association analysis using functional MUC1 variants and a nearby gene regional plot association study. A subgroup of 1686 participants was recruited for MUC1 DNA methylation analysis. After Bonferroni correction, we found that two MUC1 variants, rs4072037 and rs12411216, were significantly associated with waist circumference, systolic blood pressure, hemoglobin A1C, renal functional parameters (blood urea nitrogen, serum creatinine levels, and estimated glomerular filtration rate), albuminuria, hematocrit, hemoglobin, red blood cell count, serum uric acid level, and gout risk, with both favorable and unfavorable effects. Causal inference analysis revealed that the association between the variants and gout was partially dependent on the serum uric acid level. Both gene variants showed genome-wide significant associations with MUC1 gene-body methylation. Regional plot association analysis further revealed lead single-nucleotide polymorphisms situated at the nearby TRIM46–MUC1–THBS3–MTX1 gene region for the studied phenotypes. In conclusion, our data demonstrated the pleiotropic effects of MUC1 variants with novel associations for gout, red blood cell parameters, and MUC1 DNA methylation. These results provide further evidence in understanding the critical role of TRIM46–MUC1–THBS3–MTX1 gene region variants in the pathogenesis of cardiometabolic, renal, and hematological disorders.

Gene Body Methylation Confers Transcription Robustness in Mangroves During Long-Term Stress Adaptation.

Whether induced epigenetic changes contribute to long-term adaptation remains controversial. Recent studies indicate that environmentally cued changes in gene body methylation (gbM) can facilitate acclimatization. However, such changes are often associated with genetic variation and their contribution to long-term stress adaptation remains unclear. Using whole-genome bisulfite sequencing, we examined evolutionary gains and losses of gbM in mangroves that adapted to extreme intertidal environments. We treated mangrove seedlings with salt stress, and investigated expression changes in relation with stress-induced or evolutionarily-acquired gbM changes. Evolution and function of gbM was compared with that of genetic variation. Mangroves gained much more gbM than their terrestrial relatives, mainly through convergent evolution. Genes that convergently gained gbM during evolution are more likely to become methylated in response to salt stress in species where they are normally not marked. Stress-induced and evolutionarily convergent gains of gbM both correlate with reduction in expression variation, conferring genome-wide expression robustness under salt stress. Moreover, convergent gbM evolution is uncoupled with convergent sequence evolution. Our findings suggest that transgenerational inheritance of acquired gbM helps environmental canalization of gene expression, facilitating long-term stress adaptation of mangroves in the face of a severe reduction in genetic diversity.

GLCCI1 gene body methylation in peripheral blood is associated with asthma and asthma severity

Background and aimsEpigenetic changes play a role in the occurrence of asthma. In this study, we evaluated the methylation status of glucocorticoid-induced transcript 1 (GLCCI1) and assessed its associations with asthma and asthma severity. Materials and MethodsPeripheral blood mononuclear cells were harvested from 33 severe asthma patients, 84 mild-moderate asthma patients and 79 healthy controls of Han nationality. GLCCI1 methylation were screened using the MassArray Epityper platform (Agena). We also conducted mRNA sequencing of GLCCI1-knockout mice to further explore possible functions of this gene. ResultsWe found 5 GLCCI1 methylation sites independently correlated with asthma (adjusted p < 0.05) and perform well in asthma prediction with optimum area under the curve (AUC) value was 0.846 (p < 0.0001). In asthmatic group, only one sites independently associates with severe asthma. Area under the curve in predicting severe asthma is comparable with forced expiratory volume in 1 s predicted (AUC 0.865 and 0.857, p = 0.291). Spearman correlate analysis denoted GLCCI1 low methylation is associates with its low expression in asthma PBMCs. Its reduced level may influence PI3k-Akt and MAPK pathways by the results of RNA sequencing of GLCCI1-knockout mice (adjusted p value < 0.01). ConclusionsOur research indicates a low GLCCI1 methylation level in asthma with certain sites are lower in severe asthma group. These GLCCI1 methylation sites may be contributed to detect asthma and asthma severity.

Gene body methylation safeguards ribosomal DNA transcription by preventing PHF6-mediated enrichment of repressive histone mark H4K20me3

DNA methylation shows complex correlations with gene expression, and the role of promoter hypermethylation in repressing gene transcription has been well addressed. Emerging evidence indicates that gene body methylation promotes transcription; however, the underlying mechanisms remain to be further investigated. Here, using methylated DNA immunoprecipitation sequencing (MeDIP-seq), bisulfite genomic sequencing, and immunofluorescent labeling, we show that gene body methylation is indeed positively correlated with rRNA gene (rDNA) transcription. Mechanistically, gene body methylation is largely maintained by DNA methyltransferase 1 (DNMT1), deficiency or downregulation of which during myoblast differentiation or nutrient deprivation results in decreased gene body methylation levels, leading to increased gene body occupancy of plant homeodomain (PHD) finger protein 6 (PHF6). PHF6 binds to hypomethylated rDNA gene bodies where it recruits histone methyltransferase SUV4-20H2 to establish the repressive histone modification, H4K20me3, ultimately inhibiting rDNA transcription. These findings demonstrate that DNMT1-mediated gene body methylation safeguards rDNA transcription by preventing enrichment of repressive histone modifications, suggesting that gene body methylation serves to maintain gene expression in response to developmental and/or environmental stresses.

ASXL1 mutations are associated with distinct epigenomic alterations that lead to sensitivity to venetoclax and azacytidine

The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.

DNA methylation is not a driver of gene expression reprogramming in young honey bee workers.

The presence of DNA methylation marks within genic intervals, also called gene body methylation, is an evolutionarily-conserved epigenetic hallmark of animal and plant methylomes. In social insects, gene body methylation is thought to contribute to behavioural plasticity, for example between foragers and nurse workers, by modulating gene expression. However, recent studies have suggested that the majority of DNA methylation is sequence-specific, and therefore cannot act as a flexible mediator between environmental cues and gene expression. To address this paradox, we examined whole-genome methylation patterns in the brains and ovaries of young honey bee workers that had been subjected to divergent social contexts: the presence or absence of the queen. Although these social contexts are known to bring about extreme changes in behavioral and reproductive traits through differential gene expression, we found no significant differences between the methylomes of workers from queenright and queenless colonies. In contrast, thousands of regions were differentially methylated between colonies, and these differences were not associated with differential gene expression in the subset of genes examined. Methylation patterns were highly similar between brain and ovary tissues and only differed in nine regions. These results strongly indicate that DNA methylation is not a driver of differential gene expression between tissues or behavioral morphs. Finally, despite the lack of difference in methylation patterns, queen presence affected the expression of all four DNA methyltransferase genes, suggesting that these enzymes have roles beyond DNA methylation. Therefore, the functional role of DNA methylation in social insect genomes remains an open question.

Gene Body Methylation of the Lymphocyte-Specific Gene CARD11 Results in Its Overexpression and Regulates Cancer mTOR Signaling.

Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression. IMPLICATIONS: Our study describes a novel regulatory role of gene body DNA methylation-dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

Repression of CHROMOMETHYLASE 3 prevents epigenetic collateral damage in Arabidopsis.

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

CLLU1 Expression Is Not Confined to Chronic Lymphocytic Leukemia Cells; Putative Role of Gene-Body Hypomethylation in Regulating Splice Variant-Specific Transcriptional Dynamics

Background: Alternative splicing of the CLLU1 gene generates 6 transcript variants (accession numbers from the National Center for Biotechnology Information: AJ845162-AJ845167), two of which comprise a putative coding DNA sequence (AJ845165 and AJ845166, CDS transcripts). Previous studies showed that CLLU1 overexpression is highly confined to chronic lymphocytic leukemia (CLL) cells and that only the AJ845162 is expressed in both high- and low-risk CLL patients, while expression of the CDS-transcripts is confined to the high-risk subgroup.Pair-wise linear regression analysis revealed a powerful linear relation between the expression levels of all the splice variants, thereby it was deduced that CLLU1 transcription is orchestrated by a common promoter, which is located upstream of CLLU1 exon 1 (Buhl et al., Leukemia 2009). We recently documented (Papamichos et al., Haematologica 2016; s1) that this promoter was evolutionarily derived by an endogenous retrovirus sequence.Herein we argue that CLLU1 expression is not restricted to CLL cells while also we highlight that transcription of CDS transcripts is regulated by an alternative intronic promoter.Methods:CLLU1 high-throughput sequencing of polyadenylated RNA (RNA-Seq) data across 51 human tissues and 2 human cell lines, generated by the Genotype-Tissue Expression (GTEx) project of the US National Institutes of Health Common Fund (GTEx Analysis Release V6p), were analyzed via GTEx Portal. CLLU1 GeneChip expression data (AJ845164 transcript-specific probe set 1558185_at from Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array), generated by Genevestigator project, were downloaded from Genevisible portal.CLLU1 chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) on histone H3 protein subunit modified at lysine position 4 with trimethylation (H3K4me3) as well as transcription factor (TF) ChIP-Seq data were downloaded from the Encyclopedia of DNA Elements (ENCODE) Regulation supertrack, which is available in UCSC Genome Browser Database. Data on CLLU1 gene-body methylation status in 63 cell lines from ENCODE were downloaded from the HAIB Methyl450 ENCODE track.Results:CLLU1 GeneChip expression data show that the AJ845164 transcript (corresponding to the RP11-693J15.4 long non-coding RNA) is promiscuously expressed in various normal B-cell subpopulations andin mature B-cell neoplasms other than CLL, especially in mantle cell lymphoma.RNA-Seq data analysis reveals that AJ845162 transcript is expressed in normal somatic tissues such as skeletal muscle, salivary gland, and esophageal mucosa. Vice versa, expression of CLLU1 CDS transcripts is confined to transformed lymphocytes.Structure interrogation of CDS transcripts indicates that these variants initiate from an alternative transcriptional start site which is located 2429 nucleotides downstream from the canonical one. H3K4me3 ChIP-Seq data signify the presence of an alternative intronic promoter in this genomic segment. TF Chip-Seq data reveal that the alternative promoter region comprises high affinity binding sites for signal transducer and activator of transcription 3 (STAT3) and Activator protein 1 (AP-1). Analysis of CLLU1 -body methylation status in five lymphoblastoid cell lines reveals that the alternative promoter remains heavily methylated in these cell types. This finding is in accordance with previous studies reporting that CLLU1 gene body is hypermethylated in B-cells from healthy individuals and in IgVH-mutated (IgVH-M) CLL cases while appears hypomethylated in IgVH-unmutated (IgVH-UM) CLL cells.Conclusion: Not all CLLU1 transcripts are equal in discriminating CLL from other mature B-cell malignancies.Whilst of retroviral origin, CLLU1 upstream promoter is not subjected to stringent epigenetic repression but remains active in somatic cells, a tantalizing finding that warrants further investigation.The level of intragenic DNA methylation represents a decisive regulator of alternative promoter usage (Neri et al., Nature 2017). It would be tantalizing to speculate that CLLU1 -body hypomethylation, occuring exclusively in IgVH-unmutated CLL, is mechanistically linked to the epigenetic derepression of the alternative intronic CLLU1 promoter and the subsequent high expression of CLLU1 CDS transcripts in high-risk CLL. DisclosuresNo relevant conflicts of interest to declare.

CHH Methylation Islands: A Nonconserved Feature of Grass Genomes That Is Positively Associated with Transposable Elements but Negatively Associated with Gene-Body Methylation.

Methylated CHH (mCHH) islands are peaks of CHH methylation that occur primarily upstream to genes. These regions are actively targeted by the methylation machinery, occur at boundaries between heterochromatin and euchromatin, and tend to be near highly expressed genes. Here we took an evolutionary perspective by studying upstream mCHH islands across a sample of eight grass species. Using a statistical approach to define mCHH islands as regions that differ from genome-wide background CHH methylation levels, we demonstrated that mCHH islands are common and associate with 39% of genes, on average. We hypothesized that islands should be more frequent in genomes of large size, because they have more heterochromatin and hence more need for defined boundaries. We found, however, that smaller genomes tended to have a higher proportion of genes associated with 5′ mCHH islands. Consistent with previous work suggesting that islands reflect the silencing of the edge of transposable elements (TEs), genes with nearby TEs were more likely to have mCHH islands. However, the presence of mCHH islands was not a function solely of TEs, both because the underlying sequences of islands were often not homologous to TEs and because genic properties also predicted the presence of 5′ mCHH islands. These genic properties included length and gene-body methylation (gbM); in fact, in three of eight species, the absence of gbM was a stronger predictor of a 5′ mCHH island than TE proximity. In contrast, gene expression level was a positive but weak predictor of the presence of an island. Finally, we assessed whether mCHH islands were evolutionarily conserved by focusing on a set of 2,720 orthologs across the eight species. They were generally not conserved across evolutionary time. Overall, our data establish additional genic properties that are associated with mCHH islands and suggest that they are not just a consequence of the TE silencing machinery.

Differentially methylated gene regions between resistant and susceptible heat-phenotypes of the Pacific oyster Crassostrea gigas

Heat stress is one of the factors that affect aquaculture production of the Pacific oyster Crassostrea gigas in Mexico, and research aiming to improve heat tolerance is required. C. gigas breeding stock, produced for genetic improvement, is being implemented, and breeding lines available with 50 families were characterized for their heat-tolerance, selecting the two-most heat-resistant and the two-most heat-susceptible families. These families were analyzed for differences in their DNA methylation patterns after maintaining them for 30 days in a regime of oscillating temperatures (26 °C to 34 °C), similar to what is found in culture areas of Mexico. Whereas no differences were found in global methylation among heat-phenotypes, differentially methylated regions (DMRs) were found between phenotypes. Temperature modifies the methylation in gene bodies of C. gigas, where the heat-resistant phenotype (RR) showed more genes with DMRs in promoters (3672), exons (6340), and introns (18,685). Hyper-methylated genes among phenotypes were more abundant in RR introns (122), followed by exons (23), and promoters (2), whereas hypo-methylated regions corresponded to ten in introns, three in exons, and one in promoters. Hyper-methylated DMRs were mostly found in genes associated with processes such as regulation of gene expression, ions interactions, metabolism, and production of cellular components. This suggests that DMRs among heat-phenotypes in C. gigas families are involved in the heat-shock stress response regulated by methylation. The DMRs reported in this study could be used as methylation markers for a future broodstock selection.

Integrated transcriptomics and epigenomics reveal chamber-specific and species-specific characteristics of human and mouse hearts.

DNA methylation, chromatin accessibility, and gene expression represent different levels information in biological process, but a comprehensive multiomics analysis of the mammalian heart is lacking. Here, we applied nucleosome occupancy and methylome sequencing, which detected DNA methylation and chromatin accessibility simultaneously, as well as RNA-seq, for multiomics analysis of the 4 chambers of adult and fetal human hearts, and adult mouse hearts. Our results showed conserved region-specific patterns in the mammalian heart at transcriptome and DNA methylation level. Adult and fetal human hearts showed distinct features in DNA methylome, chromatin accessibility, and transcriptome. Novel long noncoding RNAs were identified in the human heart, and the gene expression profiles of major cardiovascular diseases associated genes were displayed. Furthermore, cross-species comparisons revealed human-specific and mouse-specific differentially expressed genes between the atria and ventricles. We also reported the relationship among multiomics and found there was a bell-shaped relationship between gene-body methylation and expression in the human heart. In general, our study provided comprehensive spatiotemporal and evolutionary insights into the regulation of gene expression in the heart.

Decoding dynamic epigenetic landscapes in human oocytes using single-cell multi-omics sequencing

Developing female human germ cells undergo genome-wide epigenetic reprogramming, but de novo DNA methylation dynamics and their interplay with chromatin states and transcriptional activation in developing oocytes is poorly understood. Here, we developed a single-cell multi-omics sequencing method, scChaRM-seq, that enables simultaneous profiling of the DNA methylome, transcriptome, and chromatin accessibility in single human oocytes and ovarian somatic cells. We observed a global increase in DNA methylation during human oocyte growth that correlates with chromatin accessibility, whereas increases of DNA methylation at specific features were associated with active transcription. Integrated analyses of multi-omics data from humans and mice revealed species-specific gene expression, and promoter accessibility contributes to gene body methylation programs. Alu elements retained low DNA methylation levels and high accessibility in early growing oocytes and were located near developmental genes in humans and mice. Together, these findings show how scChaRM-seq can provide insight into DNA methylation pattern establishment.

Transcriptomic and epigenomic remodeling occurs during vascular cambium periodicity in Populus tomentosa

Trees in temperate regions exhibit evident seasonal patterns, which play vital roles in their growth and development. The activity of cambial stem cells is the basis for regulating the quantity and quality of wood, which has received considerable attention. However, the underlying mechanisms of these processes have not been fully elucidated. Here we performed a comprehensive analysis of morphological observations, transcriptome profiles, the DNA methylome, and miRNAs of the cambium in Populus tomentosa during the transition from dormancy to activation. Anatomical analysis showed that the active cambial zone exhibited a significant increase in the width and number of cell layers compared with those of the dormant and reactivating cambium. Furthermore, we found that differentially expressed genes associated with vascular development were mainly involved in plant hormone signal transduction, cell division and expansion, and cell wall biosynthesis. In addition, we identified 235 known miRNAs and 125 novel miRNAs. Differentially expressed miRNAs and target genes showed stronger negative correlations than other miRNA/target pairs. Moreover, global methylation and transcription analysis revealed that CG gene body methylation was positively correlated with gene expression, whereas CHG exhibited the opposite trend in the downstream region. Most importantly, we observed that the number of CHH differentially methylated region (DMR) changes was the greatest during cambium periodicity. Intriguingly, the genes with hypomethylated CHH DMRs in the promoter were involved in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant–pathogen interactions during vascular cambium development. These findings improve our systems-level understanding of the epigenomic diversity that exists in the annual growth cycle of trees.

DNA methylation-mediated modulation of rapid desiccation tolerance acquisition and dehydration stress memory in the resurrection plant Boea hygrometrica.

Pre-exposure of plants to various abiotic conditions confers improved tolerance to subsequent stress. Mild drought acclimation induces acquired rapid desiccation tolerance (RDT) in the resurrection plant Boea hygrometrica, but the mechanisms underlying the priming and memory processes remain unclear. In this study, we demonstrated that drought acclimation-induced RDT can be maintained for at least four weeks but was completely erased after 18 weeks based on a combination of the phenotypic and physiological parameters. Global transcriptome analysis identified several RDT-specific rapid dehydration-responsive genes related to cytokinin and phospholipid biosynthesis, nitrogen and carbon metabolism, and epidermal morphogenesis, most of which were pre-induced by drought acclimation. Comparison of whole-genome DNA methylation revealed dehydration stress-responsive hypomethylation in the CG, CHG, and CHH contexts and acclimation-induced hypermethylation in the CHH context of the B. hygrometrica genome, consistent with the transcriptional changes in methylation pathway genes. As expected, the global promoter and gene body methylation levels were negatively correlated with gene expression levels in both acclimated and dehydrated plants but showed no association with transcriptional divergence during the procedure. Nevertheless, the promoter methylation variations in the CG and CHG contexts were significantly associated with the differential expression of genes required for fundamental genetic processes of DNA conformation, RNA splicing, translation, and post-translational protein modification during acclimation, growth, and rapid dehydration stress response. It was also associated with the dehydration stress-induced upregulation of memory genes, including pre-mRNA-splicing factor 38A, vacuolar amino acid transporter 1-like, and UDP-sugar pyrophosphorylase, which may contribute directly or indirectly to the improvement of dehydration tolerance in B. hygrometrica plants. Altogether, our findings demonstrate the potential implications of DNA methylation in dehydration stress memory and, therefore, provide a molecular basis for enhanced dehydration tolerance in plants induced by drought acclimation.

Tissue and/or developmental stage specific methylation of nrDNA in Capsicum annuum.

The nuclear ribosomal DNA (nrDNA) sequences are often used for phylogenetic analysis among organisms. Because DNA cytosine methylation and nucleolar dominancy are two common epigenetic mechanisms of nrDNA, we hypothesized that internal transcribed spacer 1 (ITS1), 5.8S rRNA and ITS2 of nrDNA sequences could be used as epigenetic biomarkers. Thus, this research was undertaken to study level and pattern of site-specific cytosine methylation of ITS1, 5.8S and ITS2 in nine tissues and/or developmental stage of pepper Capsicum annuum L. cultivar Demre Sivrisi. Tissues studied consisted of young and old roots at 30 and 90days after sowing (das), mature dry seeds and seeds at 26days of post anthesis (dpa), flowering buds at 1day before flowering, pericarps at 3, 15 and 65 dpa. Levels and patterns of DNA cytosine methylation were identified at single base resolution using bisulfite conversion sequencing. Results of this study revealed that DNA cytosine level and pattern of ITS1, 5.8S and ITS2 were different in most tissues and/or developmental stages studied. In addition, methylation levels of CG, CHG and CHH contexts were also significantly different among the regions. Based on the findings of this study, it was concluded that high level of methylation of nrDNA sequences was relatively higher as observed in transposable element and promoter. On the other hand, its tissue-specific gene expression was effective as that of gene body and promoter methylation. Overall findings revealed that methylation levels of nrDNA could be used as biomarkers for tissue identification or age estimation in plants.

Gene body methylation is under selection in Arabidopsis thaliana.

In plants, mammals and insects, some genes are methylated in the CG dinucleotide context, a phenomenon called gene body methylation (gbM). It has been controversial whether this phenomenon has any functional role. Here, we took advantage of the availability of 876 leaf methylomes in Arabidopsis thaliana to characterize the population frequency of methylation at the gene level and to estimate the site-frequency spectrum of allelic states. Using a population genetics model specifically designed for epigenetic data, we found that genes with ancestral gbM are under significant selection to remain methylated. Conversely, ancestrally unmethylated genes were under selection to remain unmethylated. Repeating the analyses at the level of individual cytosines confirmed these results. Estimated selection coefficients were small, on the order of 4 Nes = 1.4, which is similar to the magnitude of selection acting on codon usage. We also estimated that A. thaliana is losing gbM threefold more rapidly than gaining it, which could be due to a recent reduction in the efficacy of selection after a switch to selfing. Finally, we investigated the potential function of gbM through its link with gene expression. Across genes with polymorphic methylation states, the expression of gene body methylated alleles was consistently and significantly higher than unmethylated alleles. Although it is difficult to disentangle genetic from epigenetic effects, our work suggests that gbM has a small but measurable effect on fitness, perhaps due to its association to a phenotype-like gene expression.

Inference of DNA methylation patterns in molluscs.

Mollusca are the second largest and arguably most diverse phylum of the animal kingdom. This is in sharp contrast to our very limited knowledge concerning epigenetic mechanisms including DNA methylation in this invertebrate group. Here, we inferred DNA methylation patterns by analysing the normalized dinucleotide CG content in protein-coding sequences and identified DNA methyltransferases (DNMT1 and 3) in published transcriptomes and genomes of 140 species across all eight classes of molluscs. Given the evolutionary age and morphological diversity of molluscs, we expected to find evidence for diverse methylation patterns. Our inferences suggest that molluscs possess substantial levels of DNA methylation in gene bodies as a rule. Yet, we found deviations from this general picture with regard to (i) the CpG observed/expected distributions indicating a reduction in DNA methylation in certain groups and (ii) the completeness of the DNMT toolkit. Reductions were evident in Caudofoveata, Solenogastres, Polyplacophora, Monoplacophora, as well as Scaphopoda. Heterobranchia and Oegopsida were remarkable as they lacked DNMT3, usually responsible for de novo methylation, yet showed signs of DNA methylation. Our survey may serve as guidance for direct empirical analyses of DNA methylation in molluscs. This article is part of the Theo Murphy meeting issue 'Molluscan genomics: broad insights and future directions for a neglected phylum'.