Gene Body Methylation Research Articles

Evolutionary and Experimental Loss of Gene Body Methylation and Its Consequence to Gene Expression.

In flowering plants, gene body methylation (gbM) is associated with a subset of constitutively expressed genes. It has been proposed that gbM modulates gene expression. Here, we show that there are no consistent and direct differences to expression following the loss of gbM. By comparing expression of gbM genes in Arabidopsis thaliana accessions to orthologous genes in two Eutrema salsugineum genotypes, we identified both positive and negative expression differences associated with gbM loss. However, expression is largely unaffected by gbM loss in E. salsugineum. Expression differences between species were within the variation of expression observed within A. thaliana accessions that displayed variation in gbM. Furthermore, experimentally induced loss of gbM did not consistently lead to differences in expression compared to wild type. To date, there is no convincing data to support a direct causal link between the presence/absence of gbM and the modulation of expression in flowering plants.

Epimutations are associated with CHROMOMETHYLASE 3-induced de novo DNA methylation.

In many plant species, a subset of transcribed genes are characterized by strictly CG-context DNA methylation, referred to as gene body methylation (gbM). The mechanisms that establish gbM are unclear, yet flowering plant species naturally without gbM lack the DNA methyltransferase, CMT3, which maintains CHG (H = A, C, or T) and not CG methylation at constitutive heterochromatin. Here, we identify the mechanistic basis for gbM establishment by expressing CMT3 in a species naturally lacking CMT3. CMT3 expression reconstituted gbM through a progression of de novo CHG methylation on expressed genes, followed by the accumulation of CG methylation that could be inherited even following loss of the CMT3 transgene. Thus, gbM likely originates from the simultaneous targeting of loci by pathways that promote euchromatin and heterochromatin, which primes genes for the formation of stably inherited epimutations in the form of CG DNA methylation.

Interspecies association mapping links reduced CG to TG substitution rates to the loss of gene-body methylation.

Comparative genomics can unravel the genetic basis of species differences; however, successful reports on quantitative traits are still scarce. Here we present genome assemblies of 31 so-far unassembled Brassicaceae plant species and combine them with 16 previously published assemblies to establish the Brassicaceae Diversity Panel. Using a new interspecies association strategy for quantitative traits, we found a so-far unknown association between the unexpectedly high variation in CG to TG substitution rates in genes and the absence of CHROMOMETHYLASE3 (CMT3) orthologues. Low substitution rates were associated with the loss of CMT3, while species with conserved CMT3 orthologues showed high substitution rates. Species without CMT3 also lacked gene-body methylation (gbM), suggesting an evolutionary trade-off between the unknown function of gbM and low substitution rates in Brassicaceae, possibly due to low mutability of non-methylated cytosines.

Seasonal Stability and Dynamics of DNA Methylation in Plants in a Natural Environment.

DNA methylation has been considered a stable epigenetic mark but may respond to fluctuating environments. However, it is unclear how they behave in natural environments. Here, we analyzed seasonal patterns of genome-wide DNA methylation in a single clone from a natural population of the perennial Arabidopsis halleri. The genome-wide pattern of DNA methylation was primarily stable, and most of the repetitive regions were methylated across the year. Although the proportion was small, we detected seasonally methylated cytosines (SeMCs) in the genome. SeMCs in the CHH context were detected predominantly at repetitive sequences in intergenic regions. In contrast, gene-body CG methylation (gbM) itself was generally stable across seasons, but the levels of gbM were positively associated with seasonal stability of RNA expression of the genes. These results suggest the existence of two distinct aspects of DNA methylation in natural environments: sources of epigenetic variation and epigenetic marks for stable gene expression.

LMX1B mRNA expression and its gene body CpG methylation are valuable prognostic biomarkers for laryngeal squamous cell carcinoma

This study aimed to explore the prognostic value of LMX1B mRNA expression and the methylation of its CpG sites in patients with laryngeal squamous cell carcinoma (LSCC). An in-silicon analysis was performed using data from the cancer genome atlas (TCGA)-Head and Neck Squamous Carcinoma (HNSC). After screening, 112 LSCC and 10 adjacent normal tissues were identified as eligible samples for analysis. Results showed that LMX1B expression was significantly upregulated in the cancer tissues (p < 0.01) and was an independent prognostic indicator in terms of OS (HR: 1.233, 95%CI: 1.082–1.405, p = 0.002) and RFS (HR: 1.200, 95%CI: 1.002–1.438, p = 0.048). By examining the methylation profile of 55 CpG sites in LMX1B locus, we found that the promoter methylation status was irrelevant to LMX1B expression. In comparison, LMX1B expression was generally positively correlated with gene body methylation. Among the gene body CpG sites, cg13600622 methylation showed a better predictive value than LMX1B expression in terms of OS (HR: 12.363, 95%CI: 1.076–142.033, p = 0.043), while cg14204784 methylation was a better marker of shorter RFS (HR: 12.363, 95%CI: 1.076–142.033, p = 0.043). Among the known downstream genes of LMX1B, only NR4A2 expression showed a moderately negative correlation (Pearson’s r = −0.54) with it in LSCC tissues. However, this correlation was inconsistent with previous publications those reported a positive correlation between them. Based on these findings, we infer that upregulated LMX1B mRNA expression had an independent prognostic value in LSCC patients. Increased gene body methylation might be an important mechanism of its upregulation. Among the gene body CpG sites, cg13600622 and cg14204784 methylation level might be better prognostic markers than LMX1B mRNA expression in terms of OS and RFS respectively.

Abstract 479: BRAF and KRAS mutation define distinct subtypes of the CpG island methylator phenotype in colorectal cancers

Abstract BACKGROUND AND AIMS: Colorectal cancer is an epigenetically heterogeneous disease, however the extent and spectrum of the CpG Island Methylator Phenotype (CIMP) is not clear.METHODS: Genome scale methylation and transcript expression were measured using the Illumina HM450 DNA methylation and HT12 V3 expression microarrays in 216 unselected colorectal cancers. Mutations in epigenetic regulators were assessed using CIMP-classified Cancer Genome Atlas exomes.RESULTS: CIMP-High cancers dichotomised into CIMP-H1 and CIMP-H2 based on methylation profile, which was supported by over-representation of BRAF (74%, P&amp;lt;0.0001) or KRAS (55%, P&amp;lt;0.0001) mutation. Congruent with increasing methylation, there was a stepwise increase in patient age from 62 years in the CIMP-Negative subgroup to 75 years in the CIMP-H1 subgroup (P&amp;lt;0.0001). CIMP-H1 were predominantly comprised of consensus molecular subtype 1 (CMS1) cancers (70%) whilst CMS3 was over-represented in the CIMP-H2 subgroup (55%). PRC2-marked loci were subjected to significant gene body methylation in CIMP cancers (P&amp;lt;1.6x10-78). We identified oncogenes susceptible to gene body methylation and Wnt pathway antagonists resistant to gene body methylation. CIMP cluster specific mutations were observed for in chromatin remodeling genes, such as in the SWI/SNF and NuRD complexes, suggesting synthetic lethality.CONCLUSION: There are five clinically and molecularly distinct subgroups of colorectal cancer. We show a striking association between CIMP and age, gender and tumor location and identify an unidentified role for gene body methylation in progression of serrated neoplasia. These data support our recent findings that CIMP is uncommon in young patients and that BRAF mutant polyps in young patients may have limited potential for malignant progression. Citation Format: Vicki L. Whitehall, Lochlan Fennell, Troy Dumenil, Gunter Hartel, Katia Nones, Catherine Bond, Diane McKeone, Ann-Marie Patch, Stephen Kazakoff, John Pearson, Nicola Waddell, Pratyaksha Wirapati, Paul Lochead, Shuji Ogino, Sabine Tejpar, Barbara Leggett. BRAF and KRAS mutation define distinct subtypes of the CpG island methylator phenotype in colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 479.

Genetic, epigenetic and genomic effects on variation of gene expression among grape varieties.

The transcriptional regulatory structure of plant genomes is still relatively unexplored, and little is known about factors that influence expression variation in plants. We used a genetic system consisting of 10 heterozygous grape varieties with high consanguinity and high haplotypic diversity to: (i) identify regions of haplotype sharing through whole-genome resequencing and single-nucleotide polymorphism (SNP) genotyping; (ii) analyse gene expression through RNA-seq in four stages of berry development; and (iii) associate gene expression variation with genetic and epigenetic properties. We found that haplotype sharing in and around genes was positively correlated with similarity in expression and was negatively correlated with the fraction of differentially expressed genes. Genetic and epigenetic properties of the gene and the surrounding region showed significant effects on the extent of expression variation, with negative associations for the level of gene body methylation and mean expression level, and with positive associations for nucleotide diversity, structural diversity and ratio of non-synonymous to synonymous nucleotide diversity. We also observed a spatial dependency of covariation of gene expression among varieties. These results highlight relevant roles for cis-acting factors, selective constraints and epigenetic features of the gene, and the regional context in which the gene is located, in the determination of expression variation. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA385116; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA392287; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA373967 (released upon publication); https://www.ncbi.nlm.nih.gov/bioproject/PRJNA490160 (released upon publication); https://www.ncbi.nlm.nih.gov/bioproject/PRJNA265039; https://www.ncbi.nlm.nih.gov/bioproject/PRJNA265040.

Parental environments alter DNA methylation in offspring of the purple sea urchin, Strongylocentrotus purpuratus

Phenotypic plasticity, within and across generations, is a strategy by which organisms can respond rapidly to environmental change. Epigenetic modifications, such as DNA methylation, have been proposed to be involved in phenotypic plasticity. We examined the potential for the environment to mediate both transgenerational and intragenerational plasticity in DNA methylation and phenotypes in early stages of the purple sea urchin, Strongylocentrotus purpuratus, an ecologically important herbivore in kelp forest ecosystems. This approach involved a controlled laboratory experiment where adult urchins were acclimated during gametogenesis to upwelling (~1300 μatm pCO2 & 13 °C) or non-upwelling (~650 μatm pCO2 & 17 °C) conditions that are representative of their kelp forest habitat. Progeny from these adults were raised in either high (~1050 μatm) or low (~450 μatm) pCO2 treatments and sampled at three developmental stages. Differences in condition experienced by mothers were associated with differentially methylated genes in the offspring. However, differences in developmental conditions corresponded to little observable effects on gene methylation in the progeny. Variation in gene body methylation across treatments was correlated with body size of the embryos and larvae, illustrating a potential link between transgenerational phenotypic plasticity and DNA methylation. Overall, our results suggest that epigenetic factors such as DNA methylation have the potential to contribute to phenotypic plasticity in a transgenerational framework, and further, that epigenetic processes may act as a mechanism of rapid response to environmental change.

Extensive profiling in Arabidopsis reveals abundant polysome-associated 24-nt small RNAs including AGO5-dependent pseudogene-derived siRNAs.

In a reductionist perspective, plant silencing small (s)RNAs are often classified as mediating nuclear transcriptional gene silencing (TGS) or cytosolic posttranscriptional gene silencing (PTGS). Among the PTGS diagnostics is the association of AGOs and their sRNA cargos with the translation apparatus. In Arabidopsis, this is observed for AGO1 loaded with micro(mi)RNAs and, accordingly, translational-repression (TR) is one layer of plant miRNA action. Using AGO1:miRNA-mediated TR as a paradigm, we explored, with two unrelated polysome-isolation methods, which, among the ten Arabidopsis AGOs and numerous sRNA classes, interact with translation. We found that representatives of all three AGO-clades associate with polysomes, including the TGS-effector AGO4 and stereotypical 24-nt sRNAs that normally mediate TGS of transposons/repeats. Strikingly, approximately half of these annotated 24-nt siRNAs displayed unique matches in coding regions/introns of genes, and in pseudogenes, but not in transposons/repeats commonly found in their vicinity. Protein-coding gene-derived 24-nt sRNAs correlate with gene-body methylation. Those derived from pseudogenes belong to two main clusters defined by their parental-gene expression patterns, and are vastly enriched in AGO5, itself found on polysomes. Based on their tight expression pattern in developing and mature siliques, their biogenesis, and genomic/epigenomic features of their loci-of-origin, we discuss potential roles for these hitherto unknown polysome-enriched, pseudogene-derived siRNAs.

SALL3 expression balance underlies lineage biases in human induced pluripotent stem cell differentiation

Clinical applications of human induced pluripotent stem cells (hiPSCs) are expected, but hiPSC lines vary in their differentiation propensity. For efficient selection of hiPSC lines suitable for differentiation into desired cell lineages, here we identify SALL3 as a marker to predict differentiation propensity. SALL3 expression in hiPSCs correlates positively with ectoderm differentiation capacity and negatively with mesoderm/endoderm differentiation capacity. Without affecting self-renewal of hiPSCs, SALL3 knockdown inhibits ectoderm differentiation and conversely enhances mesodermal/endodermal differentiation. Similarly, loss- and gain-of-function studies reveal that SALL3 inversely regulates the differentiation of hiPSCs into cardiomyocytes and neural cells. Mechanistically, SALL3 modulates DNMT3B function and DNA methyltransferase activity, and influences gene body methylation of Wnt signaling-related genes in hiPSCs. These findings suggest that SALL3 switches the differentiation propensity of hiPSCs toward distinct cell lineages by changing the epigenetic profile and serves as a marker for evaluating the hiPSC differentiation propensity.

Comparative RNA-Sequencing and DNA Methylation Analyses of Apple (Malus domestica Borkh.) Buds with Diverse Flowering Capabilities Reveal Novel Insights into the Regulatory Mechanisms of Flower Bud Formation.

In plants, DNA methylation (i.e. chromatin modification) is important for various biological processes, including growth, development and flowering. Because 'Fuji' apple trees are alternate bearing and have a long ripening period and poor-quality flower buds, we used bud types with diverse flowering capabilities to investigate the epigenetic regulatory mechanisms influencing flower bud formation. We examined the DNA methylation changes and the transcriptional responses in the selected apple bud types. We observed that in the apple genome, approximately 79.5%, 67.4% and 23.7% of the CG, CHG and CHH sequences are methylated, respectively. For each sequence context, differentially methylated regions exhibited distinct methylation patterns among the analyzed apple bud types. Global methylation and transcriptional analyses revealed that nonexpressed genes or genes expressed at low levels were highly methylated in the gene-body regions, suggesting that gene-body methylation is negatively correlated with gene expression. Moreover, genes with methylated promoters were more highly expressed than genes with unmethylated promoters, implying promoter methylation and gene expression are positively correlated. Additionally, flowering-related genes (e.g. SOC1, AP1 and SPLs) and some transcription factor genes (e.g. GATA, bHLH, bZIP and WOX) were highly expressed in spur buds (highest flowering rate), but were associated with low methylation levels in the gene-body regions. Our findings indicate a potential correlation between DNA methylation and gene expression in apple buds with diverse flowering capabilities, suggesting an epigenetic regulatory mechanism influences apple flower bud formation.

Dynamic Cytosine DNA Methylation Patterns Associated with mRNA and siRNA Expression Profiles in Alternate Bearing Apple Trees.

Cytosine DNA methylation plays an important role in plants: it can mediate gene expression to affect plant growth and development. However, little is known about the potential involvement of cytosine DNA methylation in apple trees as well as in response to alternate bearing. Here, we performed whole-genome bisulfate sequencing to investigate genomic CG, CHG, and CHH methylation patterns, together with their global mRNA accumulation and small RNA expression in "Fuji" apple trees. Results showed that "Fuji" apple trees have a higher CHH methylation than Arabidopsis. Moreover, genomic methylation analysis revealed that CG and CHG methylation were robustly maintained at the early stage of flower induction. Additionally, differentially methylated regions (DMRs), including hypermethylated and hypomethylated DMRs, were also characterized in alternate bearing (AB) apple trees. Intriguingly, the DMRs were enriched in hormones, redox state, and starch and sucrose metabolism, which affected flowering. Further global gene expression evaluation based on methylome analysis revealed a negative correlation between gene body methylation and gene expression. Subsequent small RNA analyses showed that 24-nucleotide small interfering RNAs were activated and maintained in non-CG methylated apple trees. Our whole-genome DNA methylation analysis and RNA and small RNA expression profile construction provide valuable information for future studies.

OR24-5 Aberrant DNA Methylation in Regulatory Genomic Elements Are Associated with Invasive Behavior of Pituitary Macroadenomas: An Integrative Analysis of Epigenome-Wide Studies

A significant proportion of pituitary tumors (PT) invade adjacent structures. Few putative somatic mutations or copy number alterations have been associated with PT invasiveness. DNA methylation abnormalities in regulatory regions are known to impact gene expression and cancer behavior, but have not been fully explored in PT. Herein, we surveyed the genome-wide DNA methylation landscape of regulatory elements (promoter and enhancer) in the invasive behavior of PT. We performed a meta-analysis of genome-wide methylome profiling of invasive (Inv) and noninvasive (NInv) PT from 3 independent cohorts (32 InvPT; 35 NInvPT). Wilcoxon Rank-sum (Δβ=0.2; non-adjusted p-value <0.001) and integrative analyses using bioinformatics tools were performed. We identified 34 differentially methylated probes (DMP) in InvPT that were overall hypomethylated in relation to NInvPT. The DMPs were mainly located in distal/open sea genomic regions (OpS), enriched for enhancer regions (47%). We retrieved 6 invPT-related DMP common among the three cohorts and 4 genes previously reported to be deregulated in association with promoter and gene body methylation alterations (ITPKB; KRT12; DSC2 and LRRK1). The DMPs-associated genes were involved in myeloid cell differentiation; regulation of membrane potential and signaling by receptor tyrosine kinases. In one cohort, we also identified a few DMP in enhancer regions possibly associated with motifs predicted to bind transcription factors associated with proliferation and inflammation. Our analyses showed the association of PT invasiveness and epigenetically regulated genes as well as revealed novel candidates genes known to be therapeutic target in other tumors. These results suggest the role of distinct methylation pattern in regulatory genomic elements in the pathogenesis of invasive pituitary macroadenomas.

Cell-Free SHOX2 DNA Methylation in Blood as a Molecular Staging Parameter for Risk Stratification in Renal Cell Carcinoma Patients: A Prospective Observational Cohort Study.

Novel targeted treatments and immunotherapies have substantially changed therapeutic options for advanced and metastatic renal cell carcinomas (RCCs). However, accurate diagnostic tests for the identification of high-risk patients are urgently needed. Here, we analyzed SHOX2 mRNA expression in RCC tissues and SHOX2 gene body methylation quantitatively in circulating cell-free DNA (ccfDNA) and RCC tissues with regard to risk stratification. The clinical performance of SHOX2 methylation was tested retrospectively and prospectively in a training and testing cohort of RCC tissue samples (n = 760 in total). SHOX2 mRNA expression analysis was included in the training cohort. In matched blood plasma samples from the testing cohort (n = 100), we prospectively examined the capability of pretherapeutic quantitative SHOX2 ccfDNA methylation to assess disease stage and identify patients at high risk of death. SHOX2 gene body methylation was positively correlated with mRNA expression in RCC tissues (training cohort: Spearman ρ = 0.23, P < 0.001). SHOX2 methylation in tissue and plasma strongly correlated with an advanced disease stage (training cohort: ρ = 0.28, P < 0.001; testing cohort/tissue: ρ = 0.40, P < 0.001; testing cohort/plasma: ρ = 0.34, P = 0.001) and risk of death after initial partial or radical nephrectomy [training cohort: hazard ratio (HR) = 1.40 (95% CI, 1.24-1.57), P < 0.001; testing cohort/tissue: HR = 1.16 (95% CI, 1.07-1.27), P = 0.001; testing cohort/plasma: HR = 1.50 (95% CI, 1.29-1.74), P < 0.001]. Pretherapeutic SHOX2 ccfDNA methylation testing allows for the identification of RCC patients at high risk of death after nephrectomy. These patients might benefit from an adjuvant treatment or early initiation of a palliative treatment.

Early Parity Epigenetic Footprint of FOXA1 Gene Body in Normal Breast Tissue of Iranian Women

Background:Young age at first full-term pregnancy (FFTP) is an important factor in breast cancer risk reduction. It is postulated that this protective effect is the result of stable molecular signatures imprinted by physiological process of pregnancy, but the molecular mechanism of this protective role is unclear. The aim of the current study was to identify the effect of early FFTP on methylation status of FOXA1 gene body. FOXA1 is an essential transcription factor for mammary gland development and estrogen responsiveness of breast tissue. Methods:Fresh frozen normal breast tissues (n = 51) were collected from Iranian women who underwent cosmetic mammoplasty (27 nulliparous women and 24 parous women who have experienced first pregnancy before the age of 25). DNA was extracted and then methylated DNA immunoprecipitation (MeDIP) real-time PCR was used to assess FOXA1 gene body methylation. Results:Our results revealed that FOXA1 methylation level is significantly higher in early parous compared with nulliparous group (p = 0.041). Conclusion:Our study provides new hint about the association between early FFTP and epigenetic modifications within gene body of FOXA1 in normal breast tissue. More investigation is required for clarifying molecular mechanisms underlying this association in order to develop breast cancer prevention strategies.

Diversity of cytosine methylation across the fungal tree of life.

The generation of thousands of fungal genomes is leading to a better understanding of genes and genomic organization within the kingdom. However, the epigenome, which includes DNA and chromatin modifications, remains poorly investigated in fungi. Large comparative studies in animals and plants have deepened our understanding of epigenomic variation, particularly of the modified base 5-methylcytosine (5mC), but taxonomic sampling of disparate groups is needed to develop unifying explanations for 5mC variation. Here we utilize the largest phylogenetic resolution of 5mC methyltransferases (5mC MTases) and genome evolution to better understand levels and patterns of 5mC across fungi. We show that extant 5mC MTase genotypes are descendent from ancestral maintenance and de novo genotypes, whereas the 5mC MTases DIM-2 and RID are more recently derived, and that 5mC levels are correlated with 5mC MTase genotype and transposon content. Our survey also revealed that fungi lack canonical gene body methylation, which distinguishes fungal epigenomes from certain insect and plant species. However, some fungal species possess independently derived clusters of contiguous 5mC encompassing many genes. In some cases, DNA repair pathways and the N6-methyladenine (6mA) DNA modification negatively coevolved with 5mC pathways, which additionally contributed to interspecific epigenomic variation across fungi.

CpG traffic lights are markers of regulatory regions in human genome

BackgroundDNA methylation is involved in the regulation of gene expression. Although bisulfite-sequencing based methods profile DNA methylation at a single CpG resolution, methylation levels are usually averaged over genomic regions in the downstream bioinformatic analysis.ResultsWe demonstrate that on the genome level a single CpG methylation can serve as a more accurate predictor of gene expression than an average promoter / gene body methylation. We define CpG traffic lights (CpG TL) as CpG dinucleotides with a significant correlation between methylation and expression of a gene nearby. CpG TL are enriched in all regulatory regions. Among all promoters, CpG TL are especially enriched in poised ones, suggesting involvement of DNA methylation in their regulation. Yet, binding of only a handful of transcription factors, such as NRF1, ETS, STAT and IRF-family members, could be regulated by direct methylation of transcription factor binding sites (TFBS) or its close proximity. For the majority of TF, an alternative scenario is more likely: methylation and inactivation of the whole regulatory element indirectly represses functional TF binding with a CpG TL being a reliable marker of such inactivation.ConclusionsCpG TL provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to gene expression. CpG TL methylation can be used as reliable markers of enhancer activity and gene expression in applications, e.g. in clinic where measuring DNA methylation is easier compared to directly measuring gene expression due to more stable nature of DNA.

Single-base resolution of the methylome in sweet cherry (Prunus avium L.) during dormancy

Epigenetic modifications can provide information about the connection between genotype and phenotype and which can be directly affected by environmental conditions. In temperate perennial fruit species like sweet cherry, prolonged exposition to cold temperatures is required for dormancy release and flowering in spring. Besides genetic diversity, epigenetic regulation is believed to contribute to different chilling requirements and phenotypic plasticity among cultivars. We set out to profile methylation patterns of floral buds from sweet cherry cultivars contrasting in chilling requirement. We performed a sequence-based genome-wide differential methylation analysis using whole genome sequencing of bisulfite-treated DNA of a high chill cultivar (‘Kordia’: 1637 CH for budbreak) at four time points of cold accumulation during dormancy. As a result, we obtained global methylation levels and methylation-demethylation patterns at 0, 443, 1295 and 1637 CH. A total of 50% of the reads mapped uniquely to the reference genome of ‘Kordia’ (untreated genome). Approximately a 25-30% of total cytosines were methylated in all the conditions, corresponding to a 10-12, 9-10 and 7-8% of specific methylations at the CpG, CHG and CHH sites, respectively. Annotation of differentially methylated regions (DMRs) is under development, focusing on the distinctive patterns of transposon and gene body methylation, in an attempt to describe epigenetic marks that may be contributing to contrasting chilling requirement.

Universality of the DNA methylation codes in Eucaryotes

Genetics and epigenetics are tightly linked heritable information classes. Question arises if epigenetics provides just a set of environment dependent instructions, or whether it is integral part of an inheritance system. We argued that in the latter case the epigenetic code should share the universality quality of the genetic code. We focused on DNA methylation. Since availability of DNA methylation data is biased towards model organisms we developed a method that uses kernel density estimations of CpG observed/expected ratios to infer DNA methylation types in any genome. We show here that our method allows for robust prediction of mosaic and full gene body methylation with a PPV of 1 and 0.87, respectively. We used this prediction to complement experimental data, and applied hierarchical clustering to identify methylation types in ~150 eucaryotic species covering different body plans, reproduction types and living conditions. Our analysis indicates that there are only four gene body methylation types. These types do not follow phylogeny (i.e. phylogenetically distant clades can have identical methylation types) but they are consistent within clades. We conclude that the gene body DNA methylation codes have universality similar to the universality of the genetic code and should consequently be considered as part of the inheritance system.

Single-cell expression noise and gene-body methylation in Arabidopsis thaliana

Gene-body methylation (gbM) refers to an increased level of methylated cytosines specifically in a CG sequence context within genes. gbM is found in plant genes with intermediate expression level, which evolve slowly, and is often broadly conserved across millions of years of evolution. Intriguingly however, some plants lack gbM, and thus it remains unclear whether gbM has a function. In animals, there is support for a role of gbM in reducing erroneous transcription and transcription noise, but so far most studies in plants have tested for an effect of gbM on expression level, not noise. Here, we therefore tested whether gbM was associated with reduced expression noise in Arabidopsis thaliana, using single-cell transcriptome sequencing data from root quiescent centre cells. We find that gbM genes have lower expression noise levels than unmethylated genes. However, an analysis of covariance revealed that, if other genomic features are taken into account, this association disappears. Nonetheless, gbM genes were more consistently expressed across single-cell samples, supporting previous inference that gbM genes are constitutively expressed. Finally, we observed that fewer RNAseq reads map to introns of gbM genes than to introns of unmethylated genes, which indicates that gbM might be involved in reducing erroneous transcription by reducing intron retention.