Bisulfite Sequencing Approach Research Articles

Prenatal exposures and cell type proportions are main drivers of FKBP5 DNA methylation in maltreated and non-maltreated children

DNA methylation in peripheral tissues may be a relevant biomarker of risk for developing mental disorders after exposure to early life adversity. Genes involved in HPA axis regulation, such as FKBP5, might play a key role. In this study, we aimed to identify the main drivers of salivary FKBP5 methylation in a cohort of 162 maltreated and non-maltreated children aged 3-5 years at two measurement timepoints. We combined data from a targeted bisulfite sequencing approach for fine-mapping 49 CpGs in regulatory regions of FKBP5 and epigenetic scores for exposure to alcohol, cigarette smoke, and glucocorticoids derived from the EPIC microarray.Most variability of methylation in the FKBP5 locus was explained by estimated cell type proportions as well as epigenetic exposure scores, most prominently the glucocorticoid exposure score. While not surviving correction for multiple testing, we replicate previously reported associations of FKBP5 methylation with CM. We also detected synergistic effects of both rs1360780 genotype and the glucocorticoid exposure score on FKBP5 hypomethylation. These effects were identified in the 3’TAD, a distal regulatory of FKBP5 which is not extensively covered in Illumina arrays, emphasizing the need for fine mapping approaches. Additionally, the epigenetic glucocorticoid exposure score was associated with childhood maltreatment, maternal mental disorders, and pregnancy complications, thereby highlighting the role of glucocorticoid signaling in the epigenetic consequences of early adversity.These results underscore the need to assess cell type heterogeneity in targeted assessments of DNA methylation and show the impact of exposures beyond just childhood maltreatment such as glucocorticoid exposure.

DNA Methylation Profiling in Genetically Selected Clarias magur (Hamilton, 1822) Provides Insights into the Epigenetic Regulation of Growth and Development.

DNA methylation is an epigenetic alteration that impacts gene expression without changing the DNA sequence affecting an organism's phenotype. This study utilized a reduced representation bisulfite sequencing (RRBS) approach to investigate the patterns of DNA methylation in genetically selected Clarias magur stocks. RRBS generated 249.22 million reads, with an average of 490,120 methylation sites detected in various parts of genes, including exons, introns, and intergenic regions. A total of 896 differentially methylated regions (DMRs) were identified; 356 and 540 were detected as hyper-methylated and hypo-methylated regions, respectively. The DMRs and their association with overlapping genes were explored using whole genome data of magur, which revealed 205 genes in exonic, 210 in intronic, and 480 in intergenic regions. The analysis identified the maximum number of genes enriched in biological processes such as RNA biosynthetic process, response to growth factors, nervous system development, neurogenesis, and anatomical structure morphogenesis. Differentially methylated genes (DMGs) such as myrip, mylk3, mafb, egr3, ndnf, meis2a, foxn3, bmp1a, plxna3, fgf6, sipa1l1, mcu, cnot8, trim55b, and myof were associated with growth and development. The selected DMGs were analyzed using real-time PCR, which showed altered mRNA expression levels. This work offers insights into the epigenetic mechanisms governing growth performance regulation in magur stocks. This work provides a valuable resource of epigenetic data that could be integrated into breeding programs to select high-performing individuals.

DIAPH3 predicts survival of patients with MGMT-methylated glioblastoma.

Glioblastoma is one of the most aggressive primary brain tumors, with a poor outcome despite multimodal treatment. Methylation of the MGMT promoter, which predicts the response to temozolomide, is a well-established prognostic marker for glioblastoma. However, a difference in survival can still be detected within the MGMT methylated group, with some patients exhibiting a shorter survival than others, emphasizing the need for additional predictive factors. We analyzed DIAPH3 expression in glioblastoma samples from the cancer genome atlas (TCGA). We also retrospectively analyzed one hundred seventeen histological glioblastomas from patients operated on at Saint-Luc University Hospital between May 2013 and August 2019. We analyzed the DIAPH3 expression, explored the relationship between mRNA levels and Patient's survival after the surgical resection. Finally, we assessed the methylation pattern of the DIAPH3 promoter using a targeted deep bisulfite sequencing approach. We found that 36% and 1% of the TCGA glioblastoma samples exhibit copy number alterations and mutations in DIAPH3, respectively. We scrutinized the expression of DIAPH3 at single cell level and detected an overlap with MKI67 expression in glioblastoma proliferating cells, including neural progenitor-like, oligodendrocyte progenitor-like and astrocyte-like states. We quantitatively analyzed DIAPH3 expression in our cohort and uncovered a positive correlation between DIAPH3 mRNA level and patient's survival. The effect of DIAPH3 was prominent in MGMT-methylated glioblastoma. Finally, we report that the expression of DIAPH3 is at least partially regulated by the methylation of three CpG sites in the promoter region. We propose that combining the DIAPH3 expression with MGMT methylation could offer a better prediction of survival and more adapted postsurgical treatment for patients with MGMT-methylated glioblastoma.

MHapBrowser: a comprehensive database for visualization and analysis of DNA methylation haplotypes.

DNA methylation acts as a vital epigenetic regulatory mechanism involved in controlling gene expression. Advances in sequencing technologies have enabled characterization of methylation patterns at single-base resolution using bisulfite sequencing approaches. However, existing methylation databases have primarily focused on mean methylation levels, overlooking phased methylation patterns. The methylation status of CpGs on individual sequencing reads represents discrete DNA methylation haplotypes (mHaps). Here, we present mHapBrowser, a comprehensive database for visualizing and analyzing mHaps. We systematically processed data of diverse tissues in human, mouseand rat from public repositories, generating mHap format files for 6366 samples. mHapBrowser enables users to visualize eight mHap metrics across the genome through an integrated WashU Epigenome Browser. It also provides an online server for comparing mHap patterns across samples. Additionally, mHap files for all samples can be downloaded to facilitate local processing using downstream analysis toolkits. The utilities of mHapBrowser were demonstrated through three case studies: (i) mHap patterns are associated with gene expression; (ii) changes in mHap patterns independent of mean methylation correlate with differential expression between lung cancer subtypes; and (iii) the mHap metric MHL outperforms mean methylation for classifying tumor and normal samples from cell-free DNA. The database is freely accessible athttp://mhap.sibcb.ac.cn/.

Evaluation of DNA Methylation Profiles of LINE-1, Alu and Ribosomal DNA Repeats in Human Cell Lines Exposed to Radiofrequency Radiation.

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as "possibly carcinogenic"; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied.

Integrative multi-omics analysis depicts the methylome and hydroxymethylome in recurrent bladder cancers and identifies biomarkers for predicting PD-L1 expression

BackgroundUrinary bladder cancer (UBC) is a common malignancy of the urinary tract; however, the mechanism underlying its high recurrence and responses to immunotherapy remains unclear, making clinical outcome predictions difficult. Epigenetic alterations, especially DNA methylation, play important roles in bladder cancer development and are increasingly being investigated as biomarkers for diagnostic or prognostic predictions. However, little is known about hydroxymethylation since previous studies based on bisulfite-sequencing approaches could not differentiate between 5mC and 5hmC signals, resulting in entangled methylation results.MethodsTissue samples of bladder cancer patients who underwent laparoscopic radical cystectomy (LRC), partial cystectomy (PC), or transurethral resection of bladder tumor (TURBT) were collected. We utilized a multi-omics approach to analyze both primary and recurrent bladder cancer samples. By integrating various techniques including RNA sequencing, oxidative reduced-representation bisulfite sequencing (oxRRBS), reduced-representation bisulfite sequencing (RRBS), and whole exome sequencing, a comprehensive analysis of the genome, transcriptome, methylome, and hydroxymethylome landscape of these cancers was possible.ResultsBy whole exome sequencing, we identified driver mutations involved in the development of UBC, including those in FGFR3, KDMTA, and KDMT2C. However, few of these driver mutations were associated with the down-regulation of programmed death-ligand 1 (PD-L1) or recurrence in UBC. By integrating RRBS and oxRRBS data, we identified fatty acid oxidation-related genes significantly enriched in 5hmC-associated transcription alterations in recurrent bladder cancers. We also observed a series of 5mC hypo differentially methylated regions (DMRs) in the gene body of NFATC1, which is highly involved in T-cell immune responses in bladder cancer samples with high expression of PD-L1. Since 5mC and 5hmC alternations are globally anti-correlated, RRBS-seq-based markers that combine the 5mC and 5hmC signals, attenuate cancer-related signals, and therefore, are not optimal as clinical biomarkers.ConclusionsBy multi-omics profiling of UBC samples, we showed that epigenetic alternations are more involved compared to genetic mutations in the PD-L1 regulation and recurrence of UBC. As proof of principle, we demonstrated that the combined measurement of 5mC and 5hmC levels by the bisulfite-based method compromises the prediction accuracy of epigenetic biomarkers.

DNA methylation in transposable elements buffers the connection between three-dimensional chromatin organization and gene transcription upon rice genome duplication

IntroductionPolyploidy is a major force in plant evolution and the domestication of cultivated crops. ObjectivesThe study aimed to explore the relationship and underlying mechanism between three-dimensional (3D) chromatin organization and gene transcription upon rice genome duplication. MethodsThe 3D chromatin structures between diploid (2C) and autotetraploid (4C) rice were compared using high-throughput chromosome conformation capture (Hi-C) analysis. The study combined genetics, transcriptomics, whole-genome bisulfite sequencing (WGBS-seq) and 3D genomics approaches to uncover the mechanism for DNA methylation in modulating gene transcription through 3D chromatin architectures upon rice genome duplication. ResultsWe found that 4C rice presents weakened intra-chromosomal interactions compared to its 2C progenitor in some chromosomes. In addition, we found that changes of 3D chromatin organizations including chromatin compartments, topologically associating domains (TADs), and loops, are uncorrelated with gene transcription. Moreover, DNA methylations in the regulatory sequences of genes in compartment A/B switched regions and TAD boundaries are unrelated to their expression. Importantly, although there was no significant difference in the methylation levels in transposable elements (TEs) in differentially expressed gene (DEG) and non-DEG promoters between 2C and 4C rice, we found that the hypermethylated TEs across genes in compartment A/B switched regions and TAD boundaries may suppress the expression of these genes. ConclusionThe study proposed that the rice genome doubling might modulate TE methylation to buffer the effects of chromatin architecture on gene transcription in compartment A/B switched regions and TAD boundaries, resulting in the disconnection between 3D chromatin structure alteration and gene transcription upon rice genome duplication.

DNA methylation signatures in human neonatal blood following maternal antenatal corticosteroid treatment

Antenatal corticosteroids (ACS) are used to treat women at risk of preterm birth to improve neonatal survival. Though affected children may be at long-term risk of neurobehavioural disorders, the driving mechanisms remain unknown. Animal studies have shown that ACS exposure can lead to overlapping changes in DNA methylation between the blood and the brain, identifying gene pathways for neurodevelopment, which highlights the potential to examine peripheral blood as a surrogate for inaccessible human brain tissue. We hypothesized that differential methylation will be identified in blood of term-born neonates following ACS. Mother-infant dyads that received ACS were retrospectively identified through the Ontario Birth Study at Sinai Health Complex and matched to untreated controls for maternal age, BMI, parity and foetal sex (n = 14/group). Genome-wide methylation differences were examined at single-nucleotide resolution in DNA extracted from dried bloodspot cards using reduced representative bisulfite sequencing approaches. 505 differentially methylated CpG sites (DMCs) were identified, wherein 231 were hypermethylated and 274 were hypomethylated. These sites were annotated to 219 genes, of which USP48, SH3PXD2A, NTM, CAMK2N2, MAP6D1 were five of the top ten genes with known neurological function. Collectively, the set of hypermethylated genes were enriched for pathways of transcription regulation, while pathways of proteasome activity were enriched among the set of hypomethylated genes. This study is the first to identify DNA methylation changes in human neonatal blood following ACS. Understanding the epigenetic changes that occur in response to ACS will support future investigations to delineate the effects of prenatal glucocorticoid exposure on human development.

Location-Dependent DNA Methylation Signatures in a Clonal Invasive Crayfish.

DNA methylation is an important epigenetic modification that has been repeatedly implied in organismal adaptation. However, many previous studies that have linked DNA methylation patterns to environmental parameters have been limited by confounding factors, such as cell-type heterogeneity and genetic variation. In this study, we analyzed DNA methylation variation in marbled crayfish, a clonal and invasive freshwater crayfish that is characterized by a largely tissue-invariant methylome and negligible genetic variation. Using a capture-based subgenome bisulfite sequencing approach that covers a small, variably methylated portion of the marbled crayfish genome, we identified specific and highly localized DNA methylation signatures for specimens from geographically and ecologically distinct wild populations. These results were replicated both biologically and technically by re-sampling at different time points and by using independent methodology. Finally, we show specific methylation signatures for laboratory animals and for laboratory animals that were reared at a lower temperature. Our results thus demonstrate the existence of context-dependent DNA methylation signatures in a clonal animal.

Global analysis of DNA methylation in hepatocellular carcinoma via a whole-genome bisulfite sequencing approach

Alterations in DNA methylation patterns are considered early events in hepatocellular carcinoma (HCC). However, their mechanism and significance remain to be elucidated. We studied the genome-wide DNA methylation landscape of HCC by applying whole-genome bisulfite sequencing (WGBS) techonlogy. Overall, HCC exhibits a genome-wide hypomethylation pattern. After further annotation, we obtained 590 differentially hypermethylated genes (hyper-DMGs) and 977 differentially hypomethylated genes (hypo-DMGs) from three groups. Hyper-DMGs were mainly involved in ascorbate and alternate metabolism pathways, while hypo-DMGs were mainly involved in focal adhesion. By integrating the DMGs with HCC-related differentially expressed genes (DEGs) and DMGs from the TCGA database, we constructed prognostic model based on thirteen aberrantly methylated DEGs, and verified our prognostic model in GSE14520 dataset. This study compares the patterns of global epigenomic DNA methylation during the development of HCC, focusing on the role of DNA methylation in the early occurrence and development of HCC, providing a direction for future research on its epigenetic mechanism.

Inheritance of DNA methylation differences in the mangrove Rhizophora mangle.

The capacity to respond to environmental challenges ultimately relies on phenotypic variation which manifests from complex interactions of genetic and nongenetic mechanisms through development. While we know something about genetic variation and structure of many species of conservation importance, we know very little about the nongenetic contributions to variation. Rhizophora mangle is a foundation species that occurs in coastal estuarine habitats throughout the neotropics where it provides critical ecosystem functions and is potentially threatened by anthropogenic environmental changes. Several studies have documented landscape-level patterns of genetic variation in this species, but we know virtually nothing about the inheritance of nongenetic variation. To assess one type of nongenetic variation, we examined the patterns of DNA sequence and DNA methylation in maternal plants and offspring from natural populations of R. mangle from the Gulf Coast of Florida. We used a reduced representation bisulfite sequencing approach (epi-genotyping by sequencing; epiGBS) to address the following questions: (a) What are the levels of genetic and epigenetic diversity in natural populations of R. mangle? (b) How are genetic and epigenetic variation structured within and among populations? (c) How faithfully is epigenetic variation inherited? We found low genetic diversity but high epigenetic diversity from natural populations of maternal plants in the field. In addition, a large portion (up to ~25%) of epigenetic differences among offspring grown in common garden was explained by maternal family. Therefore, epigenetic variation could be an important source of response to challenging environments in the genetically depauperate populations of this foundation species.

Characterizing the properties of bisulfite sequencing data: maximizing power and sensitivity to identify between-group differences in DNA methylation

BackgroundThe combination of sodium bisulfite treatment with highly-parallel sequencing is a common method for quantifying DNA methylation across the genome. The power to detect between-group differences in DNA methylation using bisulfite-sequencing approaches is influenced by both experimental (e.g. read depth, missing data and sample size) and biological (e.g. mean level of DNA methylation and difference between groups) parameters. There is, however, no consensus about the optimal thresholds for filtering bisulfite sequencing data with implications for the reproducibility of findings in epigenetic epidemiology.ResultsWe used a large reduced representation bisulfite sequencing (RRBS) dataset to assess the distribution of read depth across DNA methylation sites and the extent of missing data. To investigate how various study variables influence power to identify DNA methylation differences between groups, we developed a framework for simulating bisulfite sequencing data. As expected, sequencing read depth, group size, and the magnitude of DNA methylation difference between groups all impacted upon statistical power. The influence on power was not dependent on one specific parameter, but reflected the combination of study-specific variables. As a resource to the community, we have developed a tool, POWEREDBiSeq, which utilizes our simulation framework to predict study-specific power for the identification of DNAm differences between groups, taking into account user-defined read depth filtering parameters and the minimum sample size per group.ConclusionsOur data-driven approach highlights the importance of filtering bisulfite-sequencing data by minimum read depth and illustrates how the choice of threshold is influenced by the specific study design and the expected differences between groups being compared. The POWEREDBiSeq tool, which can be applied to different types of bisulfite sequencing data (e.g. RRBS, whole genome bisulfite sequencing (WGBS), targeted bisulfite sequencing and amplicon-based bisulfite sequencing), can help users identify the level of data filtering needed to optimize power and aims to improve the reproducibility of bisulfite sequencing studies.

Epigenetic effects of parasites and pesticides on captive and wild nestling birds.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ectoparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free‐living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced‐representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we conducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identified a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontarget effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost‐effective way to characterize genome‐wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects.

Methylation-Sensitive Restriction Enzyme Quantitative Polymerase Chain Reaction Enables Rapid, Accurate, and Precise Detection of Methylation Status of the Regulatory T Cell (Treg)-Specific Demethylation Region in Primary Human Tregs.

Human regulatory T cells (Tregs) have been implicated in cancer immunotherapy and are also an emerging cellular therapeutic for the treatment of multiple indications. Although Treg stability during ex vivo culture has improved, methods to assess Treg stability such as bisulfite Sanger sequencing to determine the methylation status of the Treg-specific demethylated region (TSDR) have remained unchanged. Bisulfite Sanger sequencing is not only costly and cumbersome to perform, it is inaccurate because of relatively low read counts. Bisulfite next-generation sequencing, although more accurate, is a less accessible method. In this study, we describe the application of methylation-sensitive restriction enzymes (MSRE) and quantitative PCR (qPCR) to determine the methylation status of the TSDR. Using known ratios of Tregs and non-Tregs, we show that MSRE-qPCR can distinguish the methylation status of the TSDR in populations of cells containing increasing proportions of Tregs from 0 to 100%. In a comparison with values obtained from an established bisulfite next-generation sequencing approach for determining the methylation status of the TSDR, our MSRE-qPCR results were within 5% on average for all samples with a high percentage (>70%) of Tregs, reinforcing that MSRE-qPCR can be completed in less time than other methods with the same level of accuracy. The value of this assay was further demonstrated by quantifying differences in TSDR methylation status of Tregs treated with and without rapamycin during an ex vivo expansion culture. Together, we show that our novel application of the MSRE-qPCR to the TSDR is an optimal assay for accurate assessment of Treg purity.

DNA methylation modulates allograft survival and acute rejection after renal transplantation by regulating the mTOR pathway

Acute rejection (AR) can lead to allograft dysfunction following renal transplantation, despite immunosuppressive treatments. Accumulating evidence points out a role for epigenetic modification in immune responses. However, the mechanism and contribution of DNA methylation in allograft survival remain unclear. In this study, we followed up patients who successively experienced end‐stage renal disease, renal transplantation with allograft function or dysfunction, and hemodialysis. Peripheral blood mononuclear cells were collected at different time points for analysis of the DNA methylation. Epigenetic modifier analysis was also performed to explore its effect of methylation in a mouse model of AR. Compared with the allograft‐stable cohort, patients who experienced AR‐induced allograft dysfunction demonstrated more changes in methylation patterns. Pathway analysis revealed that the hypermethylated areas in the allograft dysfunction group were associated with genes related to the mechanistic target of rapamycin (mTOR) signaling pathway. Moreover, in the mouse AR model, treatment with the DNA methyltransferase inhibitor—decitabine regulated the Th1/2/17/regulatory T cell (Treg cell) immune response via its demethylating role in the suppressing the activity of the mTOR pathway, which ultimately ameliorated renal allograft‐related inflammatory injuries. These results revealed that changes in methylation accompany AR‐induced allograft dysfunction after renal transplantation. Epigenetics may provide new insights into predicting and improving allograft survival.

The neural stem-cell marker CD24 is specifically upregulated in IDH-mutant glioma.

BackgroundMalignant gliomas have disproportionally high morbidity and mortality. Heterozygous mutations in the isocitrate dehydrogenase 1 (IDH1) gene are most common in glioma, resulting in predominantly arginine to histidine substitution at codon 132. Because IDH1R132H requires a wild-type allele to produce (D)-2-hydroxyglutarate for epigenetic reprogramming, loss of IDH1R132H heterozygosity is associated with glioma progression in an IDH1-wildtype-like phenotype. Although previous studies have reported that transgenic IDH1R132H induces the expression of nestin—a neural stem-cell marker, the underlying mechanism remains unclear. Furthermore, this finding seems at odds with better outcome of IDH1R132H glioma because of a negative association of nestin with overall survival.MethodsGene expression was compared between IDH1R132H-hemizygous and IDH1R132H-heterozygous glioma cells under adherent and spheroid growth conditions. The results were validated for (D)-2-hydroxyglutarate responsiveness by pharmacologic agents, associations with DNA methylation by bioinformatic analysis, and associations with overall survival. Bisulfite DNA sequencing, chromatin immunoprecipitation, and pharmacological approach were used.FindingsNeural stem-cell marker genes, including CD44, NES, and PROM1, are generally downregulated in IDH-mutant gliomas and IDH1R132H-heterozygous spheroid growth compared respectively with IDH-wildtype gliomas and IDH1R132H-hemizygous spheroid growth, in agreement with their negative associations with patient outcome. In contrast, CD24 is specifically upregulated and apparently associated with better survival. CD24 and NES expression respond differentially to alteration of (D)-2-hydroxyglutarate levels. CD24 upregulation is associated with histone and DNA demethylation as opposed to hypermethylation in the downregulated genes.InterpretationThe better outcome of IDH-mutant glioma is orchestrated exquisitely through epigenetic reprogramming that directs bidirectional expression of neural stem-cell marker genes.

DNA 5-Methylcytosine-Specific Amplification and Sequencing.

DNA 5-methylcytosine (5mC)-specific mapping has been hampered by severe DNA degradation and the presence of 5-hydroxymethylcytosine (5hmC) using the conventional bisulfite sequencing approach. Here, we present a 5mC-specific whole-genome amplification method (5mC-WGA), with which we achieved 5mC retention during DNA amplification from limited input down to 10 pg scale with limited interference from 5hmC signals, providing DNA 5mC methylome with high reproducibility and accuracy.

Abstract 828: Accelerated epigenetic aging in bladder cancer patients

Abstract Aging represents the most important risk factor for many chronic diseases including cardiovascular diseases, diabetes, and cancer, therefore understanding the mechanisms of aging is a fundamental step for designing new treatments for chronic diseases. DNA methylation is the most reliable and accurate molecular marker for aging quantification, however, genome-wide DNA methylation profiling techniques, such as reduced representative bisulfite sequencing and Illumina Bead Array that are widely used in aging research are prohibitively expensive and have poor data quality at low-read coverage sites. Here we report a robust targeted bisulfite sequencing approach, called SWARM® (Simplified Whole-panel Amplification Reaction Method), for the accurate biological age determination. SWARM™ is flexible and low cost, requires relatively low DNA starting material, allows the simultaneous amplification and sequencing of hundreds of loci, and has shown to increase sample throughput. Using the SWARM® approach, we were able to analyze the methylation level of several hundreds of age-associated loci including the published Horvath Clock sites. Gender-specific age-predictive models were built using the elastic net regression of DNA methylation levels of the loci and chronological age of urine DNA samples of over 300 healthy subjects of 18 to 88 years old. Urine samples from bladder cancer patients exhibit significant age acceleration, with an average of >10 years. In brief, our gender-specific urine DNAge® analysis is a tool for the precise biological aging quantification and can be used to address questions in aging and urinary track cancers. Citation Format: Yap Ching Chew, Wei Guo, Xiaojing Yang, Paolo Piatti, Mingda Jin, Keith Booher, Benjamin Jara, Xi-Yu Jia. Accelerated epigenetic aging in bladder cancer patients [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 828.

Identification of dynamic glucocorticoid-induced methylation changes at the FKBP5 locus

BackgroundEpigenetic mechanisms may play a major role in the biological embedding of early-life stress (ELS). One proposed mechanism is that glucocorticoid (GC) release following ELS exposure induces long-lasting alterations in DNA methylation (DNAm) of important regulatory genes of the stress response. Here, we investigate the dynamics of GC-dependent methylation changes in key regulatory regions of the FKBP5 locus in which ELS-associated DNAm changes have been reported.ResultsWe repeatedly measured DNAm in human peripheral blood samples from 2 independent cohorts exposed to the GC agonist dexamethasone (DEX) using a targeted bisulfite sequencing approach, complemented by data from Illumina 450K arrays. We detected differentially methylated CpGs in enhancers co-localizing with GC receptor binding sites after acute DEX treatment (1 h, 3 h, 6 h), which returned to baseline levels within 23 h. These changes withstood correction for immune cell count differences. While we observed main effects of sex, age, body mass index, smoking, and depression symptoms on FKBP5 methylation levels, only the functional FKBP5 SNP (rs1360780) moderated the dynamic changes following DEX. This genotype effect was observed in both cohorts and included sites previously shown to be associated with ELS.ConclusionOur study highlights that DNAm levels within regulatory regions of the FKBP5 locus show dynamic changes following a GC challenge and suggest that factors influencing the dynamics of this regulation may contribute to the previously reported alterations in DNAm associated with current and past ELS exposure.

DNA methylation analysis and editing in single mammalian oocytes

Mammalian oocytes carry specific nongenetic information, including DNA methylation to the next generation, which is important for development and disease. However, evaluation and manipulation of specific methylation for both functional analysis and therapeutic purposes remains challenging. Here, we demonstrate evaluation of specific methylation in single oocytes from its sibling first polar body (PB1) and manipulation of specific methylation in single oocytes by microinjection-mediated dCas9-based targeted methylation editing. We optimized a single-cell bisulfite sequencing approach with high efficiency and demonstrate that the PB1 carries similar methylation profiles at specific regions to its sibling oocyte. By bisulfite sequencing of a single PB1, the methylation information regarding agouti viable yellow (Avy )-related coat color, as well as imprinting linked parthenogenetic development competency, in a single oocyte can be efficiently evaluated. Microinjection-based dCas9-Tet/Dnmt-mediated methylation editing allows targeted manipulation of specific methylation in single oocytes. By targeted methylation editing, we were able to reverse Avy -related coat color, generate full-term development of bimaternal mice, and correct familial Angelman syndrome in a mouse model. Our work will facilitate the investigation of specific methylation events in oocytes and provides a strategy for prevention and correction of maternally transmitted nongenetic disease or disorders.