Reduced Representation Bisulfite Sequencing Method Research Articles

Epigenome-wide association study identifies DNA methylation loci associated with handgrip strength in Chinese monozygotic twins.

Background: The decline in muscle strength and function with aging is well recognized, but remains poorly characterized at the molecular level. Here, we report the epigenetic relationship between genome-wide DNA methylation and handgrip strength (HGS) among Chinese monozygotic (MZ) twins. Methods: DNA methylation (DNAm) profiling was conducted in whole blood samples through Reduced Representation Bisulfite Sequencing method. Generalized estimating equation was applied to regress the DNAm of each CpG with HGS. The Genomic Regions Enrichment of Annotations Tool was used to perform enrichment analysis. Differentially methylated regions (DMRs) were detected using comb-p. Causal inference was performed using Inference about Causation through Examination of Familial Confounding method. Finally, we validated candidate CpGs in community residents. Results: We identified 25 CpGs reaching genome-wide significance level. These CpGs located in 9 genes, especially FBLN1, RXRA, and ABHD14B. Many enriched terms highlighted calcium channels, neuromuscular junctions, and skeletal muscle organ development. We identified 21 DMRs of HGS, with several DMRs within FBLN1, SLC30A8, CST3, and SOCS3. Causal inference indicated that the DNAm of 16 top CpGs within FBLN1, RXRA, ABHD14B, MFSD6, and TYW1B might influence HGS, while HGS influenced DNAm at two CpGs within FBLN1 and RXRA. In validation analysis, methylation levels of six CpGs mapped to FLBN1 and one CpG mapped to ABHD14B were negatively associated with HGS weakness in community population. Conclusion: Our study identified multiple DNAm variants potentially related to HGS, especially CpGs within FBLN1 and ABHD14B. These findings provide new clues to the epigenetic modification underlying muscle strength decline.

Methylome changes in Lolium perenne associated with long-term colonisation by the endophytic fungus Epichloë sp. LpTG-3 strain AR37.

Epichloë spp. often form mutualistic interactions with cool-season grasses, such as Lolium perenne. However, the molecular mechanisms underlying this interaction remain poorly understood. In this study, we employed reduced representation bisulfite sequencing method (epiGBS) to investigate the impact of the Epichloë sp. LpTG-3 strain AR37 on the methylome of L. perenne across multiple grass generations and under drought stress conditions. Our results showed that the presence of the endophyte leads to a decrease in DNA methylation across genomic features, with differentially methylated regions primarily located in intergenic regions and CHH contexts. The presence of the endophyte was consistently associated with hypomethylation in plants across generations. This research sheds new light on the molecular mechanisms governing the mutualistic interaction between Epichloë sp. LpTG-3 strain AR37 and L. perenne. It underscores the role of methylation changes associated with endophyte infection and suggests that the observed global DNA hypomethylation in L. perenne may be influenced by factors such as the duration of the endophyte-plant association and the accumulation of genetic and epigenetic changes over time.

P–055 Methylation dynamics of the sperm epigenome after chemotherapy: a case study

Abstract Study question What is the evolution of the sperm epigenome after chemotherapy in a patient with testicular cancer (TC)? Summary answer These new data on epigenetic recovery profil after TC are useful tools for counseling and reassuring these patients. What is known already An important issue for young men affected TC is how TC and its treatment will affect, transiently or permanently, their future reproductive health. The consequences of cancer treatment on the sperm epigenome during the recovery periods are topical issues of ascendant significance as epigenetic modifications to the paternal genome may have deleterious effects on the offspring. Study design, size, duration Here we report the epigenomic profiling of frozen sperm from a TC patient before and after the treatment at different time points (6, 9, 12 and 24 months) by using RRBS analysis (Reduced representation bisulfite sequencing method). Participants/materials, setting, methods A testicular tumor (testicular germ cell tumor) was diagnosed in a 30 years old patient. A cryopreservation of spermatozoa was proposed before treatment.Semen samples were obtained 2 times before treatment and 4 times after treatment (6, 9, 12 and 24 months following the initiation of treatment). Main results and the role of chance Upon collection, sampling after chemotherapy ranged from 0,6 to 4,2 million per sperm straw between 6 and 24 months after the treatment, always increasing. In order to capture the direct effect of the treatment on the methylation changes, the DMR detection has been operated between pre-chemotherapy samples (pair-wise) and the time point of 6 months. Among the 179 hqDMRs, 74 are differentially methylated between the PreCT and PostCT6m samples (16 hyper- and 68 hypo-methylated) associated with 49 DMGs (15 hyper- and 34 hypo-methylated). We further sub-clustered the 74 hqDMRs between PreCT and PostCT6m into 6 patterns, 3 hyper- and 3 hypo-methylated. Briefly, patterns P1 and P4 include hqDMRs that quickly get back to their pre-treatment methylation status just after 9th months onwards. Patterns P2 and P5 include hqDMRs that slowly get back to their pre-treatment methylation status between 12 and 24 months after treatment. Patterns P3 and P6 include hqDMRs that remain hyper- or hypo-methylated even after 24 months. We have intersected the genes (DMGs) associated with the detected hqDMRs with those known to be important or expressed during embryogenesis. We thus detected that 7 hyper-methylated and 6 hypomethylated DMGs were involved (or expressed) during embryonic / fetal development. Limitations, reasons for caution This study involves a single patient. As the patient made no major changes in his personal way of life, we hypothesized that sperm parameter variations may be attributable to the BEP treatment. Wider implications of the findings: The altered methylated status of those DMGs important for early development might modify their expression pattern and thus affect their function during key stages of embryogenesis leading to potential developmental disorders. It is important to notice that among the 110 DMGs none of them correspond to known imprinted genes. Trial registration number Not applicable

Phenotypic Response to Light Versus Shade Associated with DNA Methylation Changes in Snapdragon Plants (Antirrhinum majus).

The phenotypic plasticity of plants in response to change in their light environment, and in particularly, to shade is a schoolbook example of ecologically relevant phenotypic plasticity with evolutionary adaptive implications. Epigenetic variation is known to potentially underlie plant phenotypic plasticity. Yet, little is known about its role in ecologically and evolutionary relevant mechanisms shaping the diversity of plant populations in nature. Here we used a reference-free reduced representation bisulfite sequencing method for non-model organisms (epiGBS) to investigate changes in DNA methylation patterns across the genome in snapdragon plants (Antirrhinum majus L.). We exposed plants to sunlight versus artificially induced shade in four highly inbred lines to exclude genetic confounding effects. Our results showed that phenotypic plasticity in response to light versus shade shaped vegetative traits. They also showed that DNA methylation patterns were modified under light versus shade, with a trend towards global effects over the genome but with large effects found on a restricted portion. We also detected the existence of a correlation between phenotypic and epigenetic variation that neither supported nor rejected its potential role in plasticity. While our findings imply epigenetic changes in response to light versus shade environments in snapdragon plants, whether these changes are directly involved in the phenotypic plastic response of plants remains to be investigated. Our approach contributed to this new finding but illustrates the limits in terms of sample size and statistical power of population epigenetic approaches in non-model organisms. Pushing this boundary will be necessary before the relationship between environmentally induced epigenetic changes and phenotypic plasticity is clarified for ecologically relevant mechanisms with evolutionary implications.

Abstract 5304: Gem-interacting protein (Gmip) is potentially a new lung tumor suppressor

Abstract Introduction: We have identified a novel RASSF1C-PIWIL1-piRNA pathway that appears to promote lung cell proliferation and migration. PIWI-like proteins interact with PIWI interacting RNAs (piRNAs) to form complexes that regulate gene expression at the transcriptional and post-transcriptional levels leading to stimulation of stem cell renewal and proliferation. We previously have shown that RASSF1C regulates the expression of the PIWIL1-piRNA gene axis, suggesting the hypothesis that the RASSF1C-PIWI-piRNA pathway may promote lung cancer stem cell development and progression, in part, by epigenetically modulating the expression of growth promoting and growth inhibiting genes. To validate this hypothesis, we used a non-small cell lung cancer cell model to identify candidate genes targeted by the RASSF1C-PIWIL1-piRNA pathway through a gene methylation mechanism. Methods: We have previously conducted a study to assess the impact of over-expressing RASSF1C and knocking down RASSF1C and P1WIL1 expression on global gene DNA methylation in the NSCLC cell line H1299 using the Reduced Representation Bisulfite Sequencing method. Candidate Differentially Methylated Regions (DMR) were identified by comparing DNA methylation profiles of experimental and control cells. Statistically significant candidate genes residing in hypo- and hyper-methylated regions in lung cancer cells were identified. Results: We found that over-expression of RASSF1C and knocking down RASSF1C and PIWIL1 modulated DNA methylation of genomic regions. Among the candidate target genes identified is Gmip, a RhoA-specific GTPase-activating protein. We found over-expression of RASSF1C increases and knock-down of RASSF1C or PIWIL1 decreases intragenic methylation of Gmip. Consistent with this, RT-PCR analysis shows that Gmip mRNA levels are reduced in lung cancer cells over-expressing RASSF1C while Gmip mRNA levels are increased in cells with RASSF1C or PIWIL1 knocked down. Further, Kaplan-Meier analysis of survival of lung adenocarcinoma patients in The Cancer Genome Atlas shows that high expression of Gmip is associated with significantly higher patient survival, suggesting that Gmip may function as a tumor suppressor. Conclusion: The RASSF1C-PIWI-piRNA pathway may drive epigenome regulation and genesis of lung cancer stem cells through modulation of novel genes such as Gmip. Interestingly, Gmip is among 3781 mRNAs that are predicted to be targeted by the PIWIL1-piRNA complex in mouse male germ cells. Our findings are the first to suggest that Gmip is a human lung tumor suppressor. Since virtually nothing is known about Gmip function in human cancer, its function as a tumor suppressor in lung cancer needs to be studied and characterized. Citation Format: Yousef G. Amaar, Mark E. Reeves. Gem-interacting protein (Gmip) is potentially a new lung tumor suppressor [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 5304.

Genome-wide miRNA methylome analysis in oral cancer: possible biomarkers associated with patient survival.

The methylome associated with miRNA loci was investigated in oral cancer to explore tobacco specific methylation and potential biomarkers for patient survival. Methylome data was generated from 16 pairs of cancer-normal tissues by reduced representation bisulfite sequencing method. Differentially methylated regions were identified using the DMAP pipeline. In silico validation and Kaplan-Meier survival analyses were performed on The Cancer Genome Atlas data based on our miRNA methylome data. A total of 4310 unique differentially methylated regions, mapping to 144 miRNA loci, were identified. Three distinct groups of miRNAs were differentially methylated in cancer tissues from smokers, chewers and mixed habitués. Hypermethylation of miR-503, miR-200a/b, miR-320b and miR-489 was associated with worse 5-year survival. Differential methylation patterns in miRNA loci are associated with poor survival underscoring their potential as predictive and prognostic biomarkers in oral cancer.

Development of Enhanced Reduced Representation Bisulfite Sequencing Method for Single-cell Methylome Analysis

Single-cell analysis provides molecular signatures to define cell identity. To characterize cell types, DNA methylation patterns are often used as a flag of internal molecular status. There are a few reports of single-cell methylome techniques that involves bisulfite conversion. However, the step often causes DNA fragmentation, which leads to severe PCR substrate reduction. Here we developed a new version of single-cell reduced representation bisulfite sequencing (scRRBS) method to recover more CpG sites to be analysed. Our method succeeded to increase of 4.1 times in sample yield and 1.6 times in CpG site coverage. Importantly, our results indicate that the obtained single-cell DNA methylation sites are homogeneous among 12 cells, thus it may provide molecular barcodes for cell types. In summary, we succeed to develop enhanced scRRBS method and it will be more useful tool to define cell identity in the near future.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers.

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research.NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men.

EpiGBS: reference-free reduced representation bisulfite sequencing.

We describe epiGBS, a reduced representation bisulfite sequencing method for cost-effective exploration and comparative analysis of DNA methylation and genetic variation in hundreds of samples de novo. This method uses genotyping by sequencing of bisulfite-converted DNA followed by reliable de novo reference construction, mapping, variant calling, and distinction of single-nucleotide polymorphisms (SNPs) versus methylation variation (software is available at https://github.com/thomasvangurp/epiGBS). The output can be loaded directly into a genome browser for visualization and into RnBeads for analysis of differential methylation.