Defining context-dependent m6A RNA methylomes in Arabidopsis.

N6-Methyladenosine (m6A) prevalently occurs on cellular RNA across almost all kingdoms of life. It governs RNA fate and is essential for development and stress responses. However, the dynamic, context-dependent m6A methylomes across tissues and in response to various stimuli remain largely unknown in multicellular organisms. Here, we generate a comprehensive census that identifies m6A methylomes in 100 samples during development or following exposure to various external conditions in Arabidopsis thaliana. We demonstrate that m6A is a suitable biomarker to reflect the developmental lineage, and that various stimuli rapidly affect m6A methylomes that constitute the regulatory network required for an effective response to the stimuli. Integrative analyses of the census and its correlation with m6A regulators identify multiple layers of regulation on highly context-dependent m6A modification in response to diverse developmental and environmental stimuli, providing insights into m6A modification dynamics in the myriad contexts of multicellular organisms.

Benchmarking DNA methylation analysis of 14 alignment algorithms for whole genome bisulfite sequencing in mammals.

Whole genome bisulfite sequencing (WGBS) is an essential technique for methylome studies. Although a series of tools have been developed to overcome the mapping challenges caused by bisulfite treatment, the latest available tools have not been evaluated on the performance of reads mapping as well as on biological insights in multiple mammals. Herein, based on the real and simulated WGBS data of 14.77 billion reads, we undertook 936 mappings to benchmark and evaluate 14 wildly utilized alignment algorithms from reads mapping to biological interpretation in humans, cattle and pigs: Bwa-meth, BSBolt, BSMAP, Walt, Abismal, Batmeth2, Hisat_3n, Hisat_3n_repeat, Bismark-bwt2-e2e, Bismark-his2, BSSeeker2-bwt, BSSeeker2-soap2, BSSeeker2-bwt2-e2e and BSSeeker2-bwt2-local. Specifically, Bwa-meth, BSBolt, BSMAP, Bismark-bwt2-e2e and Walt exhibited higher uniquely mapped reads, mapped precision, recall and F1 score than other nine alignment algorithms, and the influences of distinct alignment algorithms on the methylomes varied considerably at the numbers and methylation levels of CpG sites, the calling of differentially methylated CpGs (DMCs) and regions (DMRs). Moreover, we reported that BSMAP showed the highest accuracy at the detection of CpG coordinates and methylation levels, the calling of DMCs, DMRs, DMR-related genes and signaling pathways. These results suggested that careful selection of algorithms to profile the genome-wide DNA methylation is required, and our works provided investigators with useful information on the choice of alignment algorithms to effectively improve the DNA methylation detection accuracy in mammals.

Systematic evaluation of cell-type deconvolution pipelines for sequencing-based bulk DNA methylomes.

DNA methylation analysis by sequencing is becoming increasingly popular, yielding methylomes at single-base pair and single-molecule resolution. It has tremendous potential for cell-type heterogeneity analysis using intrinsic read-level information. Although diverse deconvolution methods were developed to infer cell-type composition based on bulk sequencing-based methylomes, systematic evaluation has not been performed yet. Here, we thoroughly benchmark six previously published methods: Bayesian epiallele detection, DXM, PRISM, csmFinder+coMethy, ClubCpG and MethylPurify, together with two array-based methods, MeDeCom and Houseman, as a comparison group. Sequencing-based deconvolution methods consist of two main steps, informative region selection and cell-type composition estimation, thus each was individually assessed. With this elaborate evaluation, we aimed to establish which method achieves the highest performance in different scenarios of synthetic bulk samples. We found that cell-type deconvolution performance is influenced by different factors depending on the number of cell types within the mixture. Finally, we propose a best-practice deconvolution strategy for sequencing data and point out limitations that need to be handled. Array-based methods-both reference-based and reference-free-generally outperformed sequencing-based methods, despite the absence of read-level information. This implies that the current sequencing-based methods still struggle with correctly identifying cell-type-specific signals and eliminating confounding methylation patterns, which needs to be handled in future studies.

Comparative Epigenomics Reveals Host Diversity of the Trichinella Epigenomes and Their Effects on Differential Parasitism.

Comparative epigenomics provides new insights on evolutionary biology in relation with complex interactions between species and their environments. In the present study, we focus on deciphering the conservation and divergence of DNA methylomes during Trichinella evolution. Whole-genome bisulfite sequencing and RNA-seq were performed on the two clades of Trichinella species, in addition to whole-genome sequencing. We demonstrate that methylation patterns of sing-copy orthologous genes (SCOs) of the 12 Trichinella species are host-related and can mirror known phylogenetic relationships. Among these SCOs, we identify a panel of genes exhibiting hyper-/hypo-methylated features in gene-bodies or respective promoters that play pivotal roles in transcriptome regulation. These hyper-/hypo-methylated SCOs are also of functional significance across developmental stages, as they are highly enriched species-specific and stage-specific expressed genes both in Ad and ML stages. We further identify a set of parasitism-related functional genes that exhibit host-related differential methylation and expression among those SCOs, including p53-like transcription factor and Cdc37 that are of functional significance for elucidating differential parasitology between the two clades of Trichinella. This comparative epigenome study can help to decipher the environmental effects on differential adaptation and parasitism of the genus Trichinella.

Experimental approaches to tracking mobile genetic elements in microbial communities.

Horizontal gene transfer is an important mechanism of microbial evolution and is often driven by the movement of mobile genetic elements between cells. Due to the fact that microbes live within communities, various mechanisms of horizontal gene transfer and types of mobile elements can co-occur. However, the ways in which horizontal gene transfer impacts and is impacted by communities containing diverse mobile elements has been challenging to address. Thus, the field would benefit from incorporating community-level information and novel approaches alongside existing methods. Emerging technologies for tracking mobile elements and assigning them to host organisms provide promise for understanding the web of potential DNA transfers in diverse microbial communities more comprehensively. Compared to existing experimental approaches, chromosome conformation capture and methylome analyses have the potential to simultaneously study various types of mobile elements and their associated hosts. We also briefly discuss how fermented food microbiomes, given their experimental tractability and moderate species complexity, make ideal models to which to apply the techniques discussed herein and how they can be used to address outstanding questions in the field of horizontal gene transfer in microbial communities.

Methylome Variation Predicts Exemestane Resistance in Advanced ER+ Breast Cancer.

BACKGROUND: More than 30% of estrogen receptor-positive breast cancers are resistant to primary hormone therapy, and about 40% that initially respond to hormone therapy eventually acquire resistance. Although the mechanisms of hormone therapy resistance remain unclear, aberrant DNA methylation has been implicated in oncogenesis and drug resistance. PURPOSE: We investigated the relationship between methylome variations in circulating tumor DNA and exemestane resistance, to track hormone therapy efficacy. METHODS: We prospectively recruited 16 patients who were receiving first-line therapy in our center. All patients received exemestane-based hormone therapy after enrollment. We collected blood samples at baseline, first follow-up (after 2 therapeutic cycles) and at detection of disease progression. Disease that progressed within 6 months under exemestane treatment was considered exemestane resistance but was considered relatively exemestane-sensitive otherwise. We obtained circulating tumor DNA-derived methylomes using the whole-genome bisulfide sequencing method. Methylation calling was done by BISMARK software; differentially methylated regions for exemestane resistance were calculated afterward. RESULTS: Median follow-up for the 16 patients was 19.0 months. We found 7 exemestane resistance-related differentially methylated regions, located in different chromosomes, with both significantly different methylation density and methylation ratio. Baseline methylation density and methylation ratio of chromosome 6 [32400000-32599999] were both high in exemestane resistance. High baseline methylation ratios of chromosome 3 [67800000-67999999] (P = .013), chromosome 3 [140200000-140399999] (P = .037), and chromosome 12 [101200000-101399999] (P = .026) could also predict exemestane resistance. During exemestane treatment, synchronized changes in methylation density and methylation ratio in chromosome 6 [32400000-32599999] could accurately stratify patients in terms of progression-free survival (P = .000033). Cutoff values of methylation density and methylation ratio for chromosome 6 [149600000-149799999] were 0.066 and 0.076, respectively. CONCLUSION: Methylation change in chromosome 6 [149600000-149799999] is an ideal predictor of exemestane resistance with great clinical potential.

Whole-Genome Bisulfite Sequencing and Epigenetic Variation in Cereal Methylomes.

Whole-genome bisulfite sequencing (WGBS) is a technique used for the analysis of genome-wide DNA methylation patterns (DNA methylomes) at a single-base resolution. Here, I describe a simple DNA extraction method from rice endosperm and the universal protocol of WGBS, MethylC-sequencing library preparation. The use of benzyl chloride allows for the extraction of high-quality genomic DNA from starchy endosperm, while sodium bisulfite converts unmethylated cytosine to uracil, whereas methylated cytosine is unchanged. The bisulfite conversion of whole genome sequencing libraries before the final amplification step allows for the discrimination of methylated from unmethylated cytosines in a genome-wide manner.

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.

Methylome and transcriptome analyses of apple fruit somatic mutations reveal the difference of red phenotype.

BACKGROUND: Fruit peel colour is an important agronomic trait for fruit quality. Cytosine methylation plays an important role in gene regulation. Although the DNA methylation level of a single gene is important to affect the phenotype of mutation, there are large unknown of difference of the DNA methylation in plant and its mutants. RESULTS: Using bisulfite sequencing (BS-Seq) and RNA-sequencing (RNA-Seq), we analysed three deep-red-skinned apple (Malus × domestica) mutants (Yanfu 3, YF3; Yanfu 8, YF8; Shannonghong, SNH) and their lighter-skinned parents (Nagafu 2, NF2; Yanfu 3, YF3; Ralls, RL) to explore the different changes in methylation patterns associated with anthocyanin concentrations. We identified 13,405, 13,384, and 10,925 differentially methylated regions (DMRs) and 1987, 956, and 1180 differentially expressed genes (DEGs) in the NF2/YF3, YF3/YF8, and RL/SNH comparisons, respectively. And we found two DMR-associated DEGs involved in the anthocyanin pathway: ANS (MD06G1071600) and F3H (MD05G1074200). These genes exhibited upregulated expression in apple mutants, and differences were observed in the methylation patterns of their promoters. These results suggested that both the regulatory and structural genes may be modified by DNA methylation in the anthocyanin pathway. However, the methylation of structural genes was not the primary reason for expression-level changes. The expression of structural genes may be synergistically regulated by transcription factors and methylation changes. Additionally, the expression of the transcription factor gene MYB114 (MD17G1261100) was upregulated in the deep-red-skinned apple. CONCLUSION: Through the analysis of global methylation and transcription, we did not find the correlation between gene expression and the DNA methylation. However, we observed that the upregulated expression of ANS (MD06G1071600) and F3H (MD05G1074200) in apple mutants results in increased anthocyanin contents. Moreover, MYB114 (MD17G1261100) is likely another regulatory gene involved in apple coloration. Our data provided a new understanding about the differences in formation of apple colour mutants.

Single-base methylome analysis reveals dynamic epigenomic differences associated with water deficit in apple.

Cytosine methylation is an essential feature of epigenetic regulation and is involved in various biological processes. Although cytosine methylation has been analysed at the genomic scale for several plant species, there is a general lack of understanding of the dynamics of global and genic DNA methylation in plants growing in environments challenged with biotic and abiotic stresses. In this study, we mapped cytosine methylation at single-base resolution in the genome of commercial apple (Malus x domestica), and analysed changes in methylation patterns associated with water deficit in representative drought-sensitive and drought-tolerant cultivars. We found that the apple genome exhibits ~54%, ~38% and ~8.5% methylation at CG, CHG and CHH sequence contexts, respectively. We additionally documented changes in gene expression associated with water deficit in an attempt to link methylation and gene expression changes. Global methylation and transcription analysis revealed that promoter-unmethylated genes showed higher expression levels than promoter-methylated genes. Gene body methylation appears to be positively correlated with gene expression. Water deficit stress was associated with changes in methylation at a multitude of genes, including those encoding transcription factors (TFs) and transposable elements (TEs). These results present a methylome map of the apple genome and reveal widespread DNA methylation alterations in response to water deficit stress. These data will be helpful for understanding potential linkages between DNA methylation and gene expression in plants growing in natural environments and challenged with abiotic and biotic stresses.

Single-base resolution methylomes of upland cotton (Gossypium hirsutum L.) reveal epigenome modifications in response to drought stress.

BACKGROUND: DNA methylation, with a cryptic role in genome stability, gene transcription and expression, is involved in the drought response process in plants, but the complex regulatory mechanism is still largely unknown. RESULTS: Here, we performed whole-genome bisulfite sequencing (WGBS) and identified long non-coding RNAs on cotton leaves under drought stress and re-watering treatments. We obtained 31,223 and 30,997 differentially methylated regions (representing 2.48% of the genome) after drought stress and re-watering treatments, respectively. Our data also showed that three sequence contexts, including mCpG, mCHG, mCHH, all presented a hyper-methylation pattern under drought stress and were nearly restored to normal levels after the re-watering treatment. Among all the methylation variations, asymmetric CHH methylation was the most consistent with external environments, suggesting that methylation/demethylation in a CHH context may constitute a novel epigenetic modification in response to drought stress. Combined with the targets of long non-coding RNAs, we found that long non-coding RNAs may mediate variations in methylation patterns by splicing into microRNAs. Furthermore, the many hormone-related genes with methylation variations suggested that plant hormones might be a potential mechanism in the drought response. CONCLUSIONS: Future crop-improvement strategies may benefit by taking into account not only the DNA genetic variations in cotton varieties but also the epigenetic modifications of the genome.

The effect of cryoprotectant agents on DNA methylation patterns and progeny development in the spermatozoa of Colossoma macropomum.

DNA methylation patterns are inherited from parents and are imperative for proper embryonic development; however, alterations in these patterns can compromise fertilization and development into a fully functioning adult animal because DNA methylation is part of a complex program of gene transcription. In this study, we investigated the impact of cryoprotectant agents (CPAs) on DNA methylation patterns in spermatozoa and the consequences on embryonic development and the survival rate of progeny. Global methylation was assessed by enzymatic reactions in Colossoma macropomum spermatozoa that were cryopreserved using dimethylsulfoxide, dimethylformamide, methanol, ethyl glycol and glycerol as CPAs. Fertilization was carried out to evaluate survival rates and abnormalities in embryonic development upon treatment with each of the CPAs. Fresh semen served as the control. Our results indicated that, compared to the control group, spermatozoa cryopreservation decreased the fertilization rate and delayed embryonic development from the midblastula stage. Furthermore, spermatozoa cryopreserved in all CPAs had lower methylation levels and exhibited more delays and abnormalities during embryonic development than did fresh semen. Methanol resulted in fertilization, hatching rates and embryonic development that were closer to the control but had lower methylation levels. In conclusion, ours results show significant alterations on spermatozoa DNA methylation patterns caused by CPAs that are used in the semen cryopreservation process. DNA methylation pattern alterations affected the viability of progeny (r=0.48); however, these effects can be minimized by choosing the CPA that will compose the freezing solution.

Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome.

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types.