High-resolution methylome analysis uncovers stress-responsive genomic hotspots and drought-sensitive TE superfamilies in the clonal Lombardy poplar.

DNA methylation is environment-sensitive and can mediate stress responses. In long-lived trees, changing environments might cumulatively shape the methylome landscape over their lifetime. However, because high-resolution methylome studies usually focus on single environmental cues, it remains unclear to what extent the methylation responses are generic or stress-specific, and how this relates to their long-term stability. Here, we studied the methylome plasticity of a Populus nigra cv. 'Italica' clone that is widespread across Europe. Adult trees from a variety of geographic locations were clonally propagated in a common garden experiment, and the ramets were exposed to cold, heat, drought, herbivory, rust infection, and salicylic acid treatments. Through comprehensive whole-genome bisulfite sequencing, we analyzed stress-induced and naturally occurring DNA methylation variants. Stress-induced methylation changes predominantly targeted transposable elements. When occurring in CG/CHG contexts, the same regions were often affected by multiple stresses, suggesting a generic response of the methylome. Drought stress caused a distinct CHH hypermethylation response in transposable elements, affecting entire TE superfamilies near drought-responsive genes. Methylation differences in CG/CHG contexts that were induced by stress treatments showed striking overlap with methylation differences observed between trees from distinct geographical locations. Thus, we revealed genomic hotspots of methylation change that are not stress-specific and that contribute to natural DNA methylation variation, and we identified specific transposable element superfamilies that respond to a specific stress with possible functional consequences. Our results underscore the importance of studying the effects of multiple stressors in a single experiment for recognizing general versus stress-specific methylome responses.

The survival, gene expression, and DNA methylation of Paralichthys olivaceus impacted by the decay of green tide and bacterial infection in both laboratory and field simulation experiments.

The recurring appearance of Ulva prolifera green tides has become a pressing environmental issue, especially for marine transportation, tourism, and aquaculture in the stage of decomposition. An abundance of decaying U. prolifera leads to water acidification, hypoxia and pathogenic microorganism proliferation, threatening marine germplasm resources, particularly benthic organisms with weak escape ability. Epigenetic modification is considered to be one of the molecular mechanisms involved in the plastic adaptive response to environmental changes. However, few studies concerning the specific impact of decaying green tide on benthic animals at the epigenetic level. In this study, decomposing algal effluents of U. prolifera, sediments containing uncorrupted U. prolifera, pathogenic microorganism were considered as impact factors, to reveal the effect of decaying U. prolifera on marine economic benthic species, Paralichthys olivaceus, using both field and laboratory simulation experiments. Field simulation experiment showed higher mortality rates and serious histopathological damage than the laboratory simulation experiment. And both the decaying U. prolifera and the sediment containing U. prolifera were harmful to P. olivaceus. Genome-wide DNA methylation and transcription correlation analyses showed that the response of P. olivaceus to green tide stress and bacterial infection was mainly mediated by immune signaling pathways such as PI3K-Akt signaling pathway. DNA methylation regulates the expression of immune-related genes involved in the PI3K-Akt signaling pathway, which enables P. olivaceus to adapt to the adverse environmental stresses by resisting apoptosis. In summary, this research analyzed the potential role of P. olivaceus in decaying U. prolifera, which is of great significance for understanding the impact of decaying green tide on marine commercial fish and also provides some theoretical guidance for the proliferation and release of fish seedlings.

Novel insights into drought-induced regulation of ribosomal genes through DNA methylation in chickpea.

Modifications within the epigenome of an organism in response to external environmental conditions allow it to withstand the hostile stress factors. Drought in chickpea is a severely limiting abiotic stress factor which is known to cause huge yield loss. To analyse the methylome of chickpea in response to drought stress conditions and how it affects gene expression, we performed whole-genome bisulfite sequencing (WGBS) and RNA-seq of two chickpea genotypes which contrast for drought tolerance. It was observed that the mCHH was most variable under drought stress and the drought tolerant (DT) genotype exhibited substantial genome-wide hypomethylation as compared to the drought sensitive (DS) genotype. Specifically, there was substantial difference in gene expression and methylation for the ribosomal genes for the tolerant and sensitive genotypes. The differential expression of these genes was in complete agreement with earlier reported transcriptomes in chickpea. Many of these genes were hypomethylated (q < 0.01) and downregulated under drought stress (p < 0.01) in the sensitive genotype. The gene RPS6 (ribosomal protein small subunit) was found to be downregulated and hypomethylated in the drought sensitive genotype which could possibly lead to reduced ribosomal biosynthesis. This study provides novel insights into regulation of drought-responsive genes in chickpea.

The Change in Whole-Genome Methylation and Transcriptome Profile under Autophagy Defect and Nitrogen Starvation.

Through whole-genome bisulfite sequencing and RNA-seq, we determined the potential impact of autophagy in regulating DNA methylation in Arabidopsis, providing a solid foundation for further understanding the molecular mechanism of autophagy and how plants cope with nitrogen deficiency. A total of 335 notable differentially expressed genes (DEGs) were discovered in wild-type Arabidopsis (Col-0-N) and an autophagic mutant cultivated under nitrogen starvation (atg5-1-N). Among these, 142 DEGs were associated with hypomethylated regions (hypo-DMRs) and were upregulated. This suggests a correlation between DNA demethylation and the ability of Arabidopsis to cope with nitrogen deficiency. Examination of the hypo-DMR-linked upregulated DEGs indicated that the expression of MYB101, an ABA pathway regulator, may be regulated by DNA demethylation and the recruitment of transcription factors (TFs; ERF57, ERF105, ERF48, and ERF111), which may contribute to the growth arrest induced by abscisic acid (ABA). Additionally, we found that DNA methylation might impact the biosynthesis of salicylic acid (SA). The promoter region of ATGH3.12 (PBS3), a key enzyme in SA synthesis, was hypomethylated, combined with overexpression of PBS3 and its potential TF AT3G46070, suggesting that autophagy defects may lead to SA-activated senescence, depending on DNA demethylation. These findings suggest that DNA hypomethylation may impact the mechanism by which Arabidopsis autophagy mutants (atg5-1) respond to nitrogen deficiency, specifically in relation to ABA and SA regulation. Our evaluation of hormone levels verified that these two hormones are significantly enriched under nitrogen deficiency in atg5-1-N compared to Col-0-N.

Profiling the Epigenetic Landscape of the Spermatogonial Stem Cell: Part 2-Computational Analysis of Epigenomics Data.

The final data-generation step of genome-wide profiling of any epigenetic parameter typically involves DNA deep sequencing which yields large datasets that must then be computationally analyzed both individually and collectively to comprehensively describe the epigenetic programming that dictates cell fate and function. Here, we describe computational pipelines for analysis of bulk mepigenomic profiling data, including whole-genome bisulfite sequencing (WGBS) to detect DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to detect genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to detect genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to detect 3-dimensional interactions among distant genomic regions. In addition, we describe Chromatin State Discovery and Characterization (ChromHMM) methodology to integrate data from these individual analyses, plus that from RNA-seq analysis of gene expression, to obtain the most comprehensive overall assessment of epigenetic programming associated with gene expression.

Profiling the Epigenetic Landscape of the Spermatogonial Stem Cell-Part 1: Epigenomics Assays.

Epigenomics encompasses analyses of a variety of different epigenetic parameters which, collectively, make up the epigenetic programming that dictates cell fate and function. Here, protocols are provided for four different epigenomic methods including whole-genome bisulfite sequencing (WGBS) to assess DNA methylation patterns, chromatin immunoprecipitation-sequencing (ChIP-seq) to assess genomic patterns of either specific histone modifications or bound transcription factors, the assay for transposase-accessible chromatin-sequencing (ATAC-seq) to assess genomic patterns of chromatin accessibility, and high-throughput chromosome conformation capture-sequencing (Hi-C-seq) to assess three-dimensional interactions among distant genomic regions, plus computational methodology to integrate data from those four methodologies using Chromatin State Discovery and Characterization (ChromHMM) to obtain the most comprehensive overall assessment of epigenetic programming.

Post-bisulfite Adaptor Tagging Based on an ssDNA Ligation Technique (tPBAT).

Post-bisulfite adaptor tagging (PBAT) is a concept that enables the preparation of an efficient sequencing library from bisulfite-treated DNA, and it also means the protocol implemented the concept. Although the previous PBAT or rPBAT was sensitive enough for single-cell methylome analysis, the protocol had several drawbacks owing to the repeated random priming reactions. To resolve these problems, we developed a unique single-strand DNA ligation technique, termed TACS ligation, and established a new protocol called tPBAT. With tPBAT, the data quality improved, with a longer insert and higher mapping rate than that obtained with rPBAT. In addition, paired-end sequencing and indexing were supported by the default. In this chapter, the tPBAT protocol is introduced, and a thorough description of its application to small samples is provided.

Post-bisulfite Adaptor Tagging with a Highly Efficient Single-Stranded DNA Ligation Technique.

Post-bisulfite adaptor tagging (PBAT) is a procedure for efficiently preparing a sequencing library for whole-genome bisulfite sequencing (WGBS). The original version of the PBAT protocol was highly efficient, such that it helped realize library preparation from samples of limited amounts. However, two rounds of random priming reactions employed in the original protocol limited further improvement of the PBAT protocol in terms of read length and mapping rate. In this chapter, an improved version of the PBAT protocol called tPBAT is described.

Genome-wide differential DNA methylation analysis of MDA-MB-231 breast cancer cells treated with curcumin derivatives, ST08 and ST09.

ST08 and ST09 are potent curcumin derivatives with antiproliferative, apoptotic, and migrastatic properties. Both ST08 and ST09 exhibit in vitro and in vivo anticancer properties. As reported earlier, these derivatives were highly cytotoxic towards MDA-MB-231 triple-negative breast cancer cells with IC50 values in the nanomolar (40-80nM) range.In this study,we performed whole-genome bisulfite sequencing(WGBS) of untreated (control), ST08 and ST09 (treated) triple-negative breast cancer cell line MDA-MB-231 to unravel epigenetic changes induced by the drug. We identified differentially methylated sites (DMSs) enriched in promoter regions across the genome. Analysis of the CpG island promoter methylation identified 12 genes common to both drugs, and 50% of them are known to be methylated in patient samples that were hypomethylated by drugs belonging to the homeobox family transcription factors.Methylation analysis of the gene body revealed 910 and 952 genes to be hypermethylatedin ST08 and ST09 treated MDA-MB-231 cells respectively. Correlation of the gene body hypermethylation with expression revealed CACNAH1 to be upregulated in ST08 treatment and CDH23 upregulation in ST09.Further, integrated analysis of the WGBS with RNA-seq identified uniquely altered pathways - ST08 altered ECM pathway, and ST09 cell cycle, indicating drug-specific signatures.

Fine mapping and characterization of RLL6 locus required for anti-silencing of a transgene and DNA demethylation in Arabidopsis thaliana.

DNA methylation patterns in plants are dynamically shaped by the antagonistic actions of DNA methylation and demethylation pathways. Although the DNA methylation pathway has been well studied, the DNA demethylation pathway, however, are not fully understood so far. To gain deeper insights into the mechanisms of DNA demethylation pathway, we conducted a genetic screening for proteins that were involved in preventing epigenetic gene silencing, and then the ones, which were also implicated in DNA demethylation pathway, were used for further studies. Eventually, a mutant with low luciferase luminescence (low LUC luminescence) was recovered, and named reduced LUC luminescence 6-1 (rll6-1). Map-based cloning revealed that rll6-1 mutation was located on chromosome 4, and there were a total of 10 candidate genes residing within such a region. Analyses of genome-wide methylation patterns of rll6-1 mutant showed that mutation of RLL6 locus led to 3,863 hyper-DMRs (DMRs for differentially methylated regions) throughout five Arabidopsis chromosomes, and elevated DNA methylation level of 2 × 35S promoter, which was similar to that found in the ros1 (repressor of silencing 1) mutant. Further analysis demonstrated that there were 1,456 common hyper-DMRs shared by rll6-1 and ros1-7 mutants, suggesting that both proteins acted together in a synergistic manner to remove DNA methylation. Further investigations demonstrated that mutation of RLL6 locus did not affect the expression of the four genes of the DNA glycosylase/lyase family. Thus, our results demonstrate that RLL6 locus-encoded protein not only participates in transcriptional anti-silencing of a transgene, but is also involved in DNA demethylation pathway.

Comparative DNA Methylome of Phytoplasma Associated Retrograde Metamorphosis in Sesame (Sesamum indicum L.).

Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. The mechanism of the dramatic conversion of flowers to leafy structures leading to yield losses and the drastic reduction in leaf size due to Phytoplasma infection remains yet to be identified. Cytosine methylation profiles of healthy and infected sesame plants studied using Whole Genome Bisulfite Sequencing (WGBS) and Quantitative analysis of DNA methylation with the real-time PCR (qAMP) technique revealed altered DNA methylation patterns upon infection. Phyllody was associated with global cytosine hypomethylation, though predominantly in the CHH (where H = A, T or C) context. Interestingly, comparable cytosine methylation levels were observed between healthy and little leaf-affected plant samples in CG, CHG and CHH contexts. Among the different genomic fractions, the highest number of differentially methylated Cytosines was found in the intergenic regions, followed by promoter, exonic and intronic regions in decreasing order. Further, most of the differentially methylated genes were hypomethylated and were mainly associated with development and defense-related processes. Loci for STOREKEEPER protein-like, a DNA-binding protein and PP2-B15, an F-Box protein, responsible for plugging sieve plates to maintain turgor pressure within the sieve tubes were found to be hypomethylated by WGBS, which was confirmed by methylation-dependent restriction digestion and qPCR. Likewise, serine/threonine-protein phosphatase-7 homolog, a positive regulator of cryptochrome signaling involved in hypocotyl and cotyledon growth and probable O-methyltransferase 3 locus were determined to be hypermethylated. Phytoplasma infection-associated global differential methylation as well as the defense and development-related loci reported here for the first time significantly elucidate the mechanism of phytoplasma-associated disease development.

From Methylome to Integrative Analysis of Tissue Specificity.

DNA methylation is the most studied epigenetic mark in both plants and animals. The gold standard for assaying genome-wide DNA methylation at single-base resolution is whole-genome bisulfite sequencing (WGBS). Here, we describe an improved procedure for WGBS and original bioinformatic workflows applied to unravel tissue-specific variations of the methylome in relation to gene expression and accumulation of secondary metabolites in the medicinal plant Catharanthus roseus.

Multiomics analysis of male infertility†.

Infertility affects 8-12% of couples globally, and the male factor is a primary cause in ~50% of couples. Male infertility is a multifactorial reproductive disorder, which can be caused by paracrine and autocrine factors, hormones, genes, and epigenetic changes. Recent studies in rodents and most notably in humans using multiomics approach have yielded important insights into understanding the biology of spermatogenesis. Nonetheless, the etiology and pathogenesis of male infertility are still largely unknown. In this review, we summarized and critically evaluated findings based on the use of advanced technologies to compare normal and obstructive azoospermic versus nonobstructive azoospermic men, including whole-genome bisulfite sequencing, single-cell RNA-seq, whole-exome sequencing, and transposase-accessible chromatin using sequencing. It is obvious that the multiomics approach is the method of choice for basic research and clinical studies including clinical diagnosis of male infertility.

Phenotypic and Methylome Responses to Salt Stress in Arabidopsis thaliana Natural Accessions.

Salt stress threatens plant growth, development and crop yields, and has become a critical global environmental issue. Increasing evidence has suggested that the epigenetic mechanism such as DNA methylation can mediate plant response to salt stress through transcriptional regulation and transposable element (TE) silencing. However, studies exploring genome-wide methylation dynamics under salt stress remain limited, in particular, for studies on multiple genotypes. Here, we adopted four natural accessions of the model species Arabidopsis thaliana and investigated the phenotypic and genome-wide methylation responses to salt stress through whole-genome bisulfite sequencing (WGBS). We found that salt stress significantly changed plant phenotypes, including plant height, rosette diameter, fruit number, and aboveground biomass, and the change in biomass tended to depend on accessions. Methylation analysis revealed that genome-wide methylation patterns depended primarily on accessions, and salt stress caused significant methylation changes in ∼ 0.1% cytosines over the genomes. About 33.5% of these salt-induced differential methylated cytosines (DMCs) were located to transposable elements (TEs). These salt-induced DMCs were mainly hypermethylated and accession-specific. TEs annotated to have DMCs (DMC-TEs) across accessions were found mostly belonged to the superfamily of Gypsy, a type II transposon, indicating a convergent DMC dynamic on TEs across different genetic backgrounds. Moreover, 8.0% of salt-induced DMCs were located in gene bodies and their proximal regulatory regions. These DMCs were also accession-specific, and genes annotated to have DMCs (DMC-genes) appeared to be more accession-specific than DMC-TEs. Intriguingly, both accession-specific DMC-genes and DMC-genes shared by multiple accessions were enriched in similar functions, including methylation, gene silencing, chemical homeostasis, polysaccharide catabolic process, and pathways relating to shifts between vegetative growth and reproduction. These results indicate that, across different genetic backgrounds, methylation changes may have convergent functions in post-transcriptional, physiological, and phenotypic modulation under salt stress. These convergent methylation dynamics across accession may be autonomous from genetic variation or due to convergent genetic changes, which requires further exploration. Our study provides a more comprehensive picture of genome-wide methylation dynamics under salt stress, and highlights the importance of exploring stress response mechanisms from diverse genetic backgrounds.

Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human-Arabidopsis Hybrid Cell Line.

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human-Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.

Changes in DNA Methylation in Arabidopsis thaliana Plants Exposed Over Multiple Generations to Gamma Radiation.

Previous studies have found indications that exposure to ionising radiation (IR) results in DNA methylation changes in plants. However, this phenomenon is yet to be studied across multiple generations. Furthermore, the exact role of these changes in the IR-induced plant response is still far from understood. Here, we study the effect of gamma radiation on DNA methylation and its effect across generations in young Arabidopsis plants. A multigenerational set-up was used in which three generations (Parent, generation 1, and generation 2) of 7-day old Arabidopsis thaliana plants were exposed to either of the different radiation treatments (30, 60, 110, or 430 mGy/h) or to natural background radiation (control condition) for 14 days. The parental generation consisted of previously non-exposed plants, whereas generation 1 and generation 2 plants had already received a similar irradiation in the previous one or two generations, respectively. Directly after exposure the entire methylomes were analysed with UPLC-MS/MS to measure whole genome methylation levels. Whole genome bisulfite sequencing was used to identify differentially methylated regions (DMRs), including their methylation context in the three generations and this for three different radiation conditions (control, 30 mGy/h, and 110 mGy/h). Both intra- and intergenerational comparisons of the genes and transposable elements associated with the DMRs were made. Taking the methylation context into account, the highest number of changes were found for cytosines followed directly by guanine (CG methylation), whereas only limited changes in CHG methylation occurred and no changes in CHH methylation were observed. A clear increase in IR-induced DMRs was seen over the three generations that were exposed to the lowest dose rate, where generation 2 had a markedly higher number of DMRs than the previous two generations (Parent and generation 1). Counterintuitively, we did not see significant differences in the plants exposed to the highest dose rate. A large number of DMRs associated with transposable elements were found, the majority of them being hypermethylated, likely leading to more genetic stability. Next to that, a significant number of DMRs were associated with genes (either in their promoter-associated region or gene body). A functional analysis of these genes showed an enrichment for genes related to development as well as various stress responses, including DNA repair, RNA splicing, and (a)biotic stress responses. These observations indicate a role of DNA methylation in the regulation of these genes in response to IR exposure and shows a possible role for epigenetics in plant adaptation to IR over multiple generations.

Whole-genome methylation analysis reveals novel epigenetic perturbations of congenital scoliosis.

Congenital scoliosis (CS) is a congenital disease caused by malformations of vertebrae. Recent studies demonstrated that DNA modification could contribute to the pathogenesis of disease. This study aims to identify epigenetic perturbations that may contribute to the pathogenesis of CS. Four CS patients with hemivertebra were enrolled and underwent spine correction operations. DNA was extracted from the hemivertebrae and spinal process collected from the specimen during the hemivertebra resection. Genome-wide DNA methylation profiling was examined at base-pair resolution using whole-genome bisulfite sequencing (WGBS). We identified 343 genes with hyper-differentially methylated regions (DMRs) and 222 genes with hypo-DMRs, respectively. These genes were enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, calcium signaling pathway, and axon guidance in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and were enriched in positive regulation of cell morphogenesis involved in differentiation, regulation of cell morphogenesis involved in differentiation, and regulation of neuron projection development in Biological Process of Gene Ontology (GO-BP) terms. Hyper-DMR-related genes, including IGHG1, IGHM, IGHG3, RNF213, and GSE1, and hypo DMR-related genes, including SORCS2, COL5A1, GRID1, RGS3, and ROBO2, may contribute to the pathogenesis of hemivertebra. The aberrant DNA methylation may be associated with the formation of hemivertebra and congenital scoliosis.

Myxosporea (Myxozoa, Cnidaria) Lack DNA Cytosine Methylation.

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.

BCREval: a computational method to estimate the bisulfite conversion ratio in WGBS.

BACKGROUND: Whole genome bisulfite sequencing (WGBS) also known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. One of the key steps in the assay is converting unmethylated cytosines into thymines (BS conversion). Incomplete conversion of unmethylated cytosines can introduce false positive methylation call. Developing a quick method to evaluate bisulfite conversion ratio (BCR) is benefit for both quality control and data analysis of WGBS. RESULTS: Here we provide a computational method named "BCREval" to estimate the unconverted rate (UCR) by using telomeric repetitive DNA as native spike-in control. We tested the method by using public WGBS data and found that it is very stable and most of BS conversion assays can achieve> 99.5% efficiency. The non-CpG DNA methylation at telomere fits a binomial model and may result from a random process with very low possibility (the ratio < 0.4%). And the comparison between BCREval and Bismark (Krueger and Andrews, Bioinformatics 27:1571-1572, 2011), a widely used BCR evaluator, suggests that our algorithm is much faster and more efficient than the latter. CONCLUSION: Our method is a simple but robust method to QC and speculates BCR for WGBS experiments to make sure it achieves acceptable level. It is faster and more efficient than current tools and can be easily integrated into presented WGBS pipelines.

Investigation of Gene Expression and DNA Methylation From Seven Different Brain Regions of a Crab-Eating Monkey as Determined by RNA-Seq and Whole-Genome Bisulfite Sequencing.

The crab-eating monkey is widely used in biomedical research for pharmacological experiments. Epigenetic regulation in the brain regions of primates involves complex patterns of DNA methylation. Previous studies of methylated CpG-binding domains using microarray technology or peak identification of sequence reads mostly focused on developmental stages or disease, rather than normal brains. To identify correlations between gene expression and DNA methylation levels that may be related to transcriptional regulation, we generated RNA-seq and whole-genome bisulfite sequencing data from seven different brain regions from a single crab-eating monkey. We identified 92 genes whose expression levels were significantly correlated, positively or negatively, with DNA methylation levels. Among them, 11 genes exhibited brain region-specific characteristics, and their expression patterns were strongly correlated with DNA methylation level. Nine genes (SLC2A5, MCM5, DRAM1, TTC12, DHX40, COR01A, LRAT, FLVCR2, and PTER) had effects on brain and eye function and development, and two (LHX6 and MEST) were previously identified as genes in which DNA methylation levels change significantly in the promoter region and are therefore considered brain epigenetic markers. Furthermore, we characterized DNA methylation of repetitive elements at the whole genome through repeat annotation at single-base resolution. Our results reveal the diverse roles of DNA methylation at single-base resolution throughout the genome and reflect the epigenetic variations in adult brain tissues.