scholarly journals Perinatal exposure to lead results in altered DNA methylation in adult mouse liver and blood: Implications for target versus surrogate tissue use in environmental epigenetics

2019 ◽  
Author(s):  
LK Svoboda ◽  
K Neier ◽  
R Cavalcante ◽  
Z Tsai ◽  
TR Jones ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Epigenetic Mechanism ◽  
Perinatal Exposure ◽  
Bioinformatics Pipeline ◽  
Reduced Representation ◽  
Environmental Health Sciences ◽  
Reduced Representation Bisulfite Sequencing ◽  
Pb Exposure ◽  
Surrogate Tissues ◽  
And Control

ABSTRACTBackgroundDNA methylation is a critical epigenetic mechanism linking early developmental environment to long-term health. In humans, the extent to which toxicant-induced changes in DNA methylation in surrogate tissues, such as blood, mirror those in the target tissues is unclear. The Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET II) consortium was established by the National Institute of Environmental Health Sciences to address the utility of surrogate tissues as proxies for toxicant-induced epigenetic changes in target tissues.ObjectivesThe objective of this study was to investigate the effects of perinatal exposure to a human environmentally relevant level (32 ppm in maternal drinking water) of lead (Pb) on liver and blood DNA methylation in adult male and female mice. We hypothesized that developmental Pb exposure would lead to persistent changes in DNA methylation, and that a subset of differentially methylated loci would overlap between liver and blood.MethodsEnhanced reduced-representation bisulfite sequencing was used to assess DNA methylation in 5 month old Pb-exposed and control mice. Sex-stratified modeling of differential methylation by Pb exposure was conducted using an established bioinformatics pipeline.ResultsAlthough Pb exposure ceased at 3 weeks of age, we observed thousands of stably modified, sex-specific differentially methylated regions in the blood and liver of Pb-exposed animals, including 44 genomically imprinted loci. In males, we discovered 5 sites that overlapped between blood and liver, and exhibited changes in DNA methylation in the same direction in both tissues.ConclusionsThese data demonstrate that perinatal exposure to Pb induces sex-specific changes in hepatic DNA methylation in adulthood, some of which are also present in blood. Ongoing studies will provide additional exposure-specific insights, and include other epigenetic marks that will enable further refinement of the design and analysis of human studies where target tissues are inaccessible.

scholarly journals Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Aniruddha Chatterjee ◽  
Euan J. Rodger ◽  
Peter A. Stockwell ◽  
Robert J. Weeks ◽  
Ian M. Morison
Keyword(s):  
Bisulfite Sequencing ◽  
Flow Cell ◽  
Throughput Capacity ◽  
Sequencing Data ◽  
Bioinformatics Pipeline ◽  
Illumina Hiseq ◽  
Reduced Representation ◽  
Reduced Representation Bisulfite Sequencing ◽  
Unique Mapping ◽  
Nucleotide Resolution

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background.

scholarly journals Characterization of Global DNA Methylation in Different Gene Regions Reveals Candidate Biomarkers in Pigs with High and Low Levels of Boar Taint

2020 ◽  
Vol 7 (2) ◽  
pp. 77 ◽  
Author(s):  
Xiao Wang ◽  
Haja N. Kadarmideen
Keyword(s):  
Dna Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Gene Interaction ◽  
Boar Taint ◽  
Reduced Representation ◽  
Gene Interaction Networks ◽  
Reduced Representation Bisulfite Sequencing ◽  
Pathways Analysis ◽  
Porcine Gene

DNA methylation of different gene components, including different exons and introns, or different lengths of exons and introns is associated with differences in gene expression. To investigate the methylation of porcine gene components associated with the boar taint (BT) trait, this study used reduced representation bisulfite sequencing (RRBS) data from nine porcine testis samples in three BT groups (low, medium and high BT). The results showed that the methylation levels of the first exons and first introns were lower than those of the other exons and introns. The first exons/introns of CpG island regions had even lower levels of methylation. A total of 123 differentially methylated promoters (DMPs), 194 differentially methylated exons (DMEs) and 402 differentially methylated introns (DMIs) were identified, of which 80 DMPs (DMP-CpGis), 112 DMEs (DME-CpGis) and 166 DMIs (DMI-CpGis) were discovered in CpG islands. Importantly, GPX1 contained one each of DMP, DME, DMI, DMP-CpGi, DME-CpGi and DMI-CpGi. Gene-GO term relationships and pathways analysis showed DMP-CpGi-related genes are mainly involved in methylation-related biological functions. In addition, gene–gene interaction networks consisted of nodes that were hypo-methylated GPX1, hypo-methylated APP, hypo-methylated ATOX1, hyper-methylated ADRB2, hyper-methylated RPS6KA1 and hyper-methylated PNMT. They could be used as candidate biomarkers for reducing boar taint in pigs, after further validation in large cohorts.

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

2011 ◽  
Vol 6 (4) ◽  
pp. 468-481 ◽  
Author(s):  
Hongcang Gu ◽  
Zachary D Smith ◽  
Christoph Bock ◽  
Patrick Boyle ◽  
Andreas Gnirke ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Reduced Representation ◽  
Reduced Representation Bisulfite Sequencing ◽  
Dna Methylation Profiling ◽  
Genome Scale ◽  
Methylation Profiling

scholarly journals DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rajbir Nath Batra ◽  
Aviezer Lifshitz ◽  
Ana Tufegdzic Vidakovic ◽  
Suet-Feung Chin ◽  
Ankita Sati-Batra ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cpg Island ◽  
Cpg Islands ◽  
Tumor Grade ◽  
Normal Breast ◽  
Breast Cancers ◽  
Reduced Representation ◽  
Reduced Representation Bisulfite Sequencing ◽  
Transcriptional Changes ◽  
In Cis

AbstractDNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.

scholarly journals On the Use of Blood Samples for Measuring DNA Methylation in Ecological Epigenetic Studies

2020 ◽  
Vol 60 (6) ◽  
pp. 1558-1566 ◽  
Author(s):  
Arild Husby
Keyword(s):  
Dna Methylation ◽  
Phenotypic Variation ◽  
Evolutionary Biology ◽  
Phenotypic Diversity ◽  
Genomic Region ◽  
Epigenetic Mechanism ◽  
Blood Samples ◽  
Reduced Representation ◽  
Bisulfite Treatment ◽  
Methylation Patterns

Synopsis There is increasing interest in understanding the potential for epigenetic factors to contribute to phenotypic diversity in evolutionary biology. One well studied epigenetic mechanism is DNA methylation, the addition of a methyl group to cytosines, which have the potential to alter gene expression depending on the genomic region in which it takes place. Obtaining information about DNA methylation at genome-wide scale has become straightforward with the use of bisulfite treatment in combination with reduced representation or whole-genome sequencing. While it is well recognized that methylation is tissue specific, a frequent limitation for many studies is that sampling-specific tissues may require sacrificing individuals, something which is generally undesirable and sometimes impossible. Instead, information about DNA methylation patterns in the blood is frequently used as a proxy tissue. This can obviously be problematic if methylation patterns in the blood do not reflect that in the relevant tissue. Understanding how, or if, DNA methylation in blood reflect DNA methylation patterns in other tissues is therefore of utmost importance if we are to make inferences about how observed differences in methylation or temporal changes in methylation can contribute to phenotypic variation. The aim of this review is to examine what we know about the potential for using blood samples in ecological epigenetic studies. I briefly outline some methods by which we can measure DNA methylation before I examine studies that have compared DNA methylation patterns across different tissues and, finally, examine how useful blood samples may be for ecological studies of DNA methylation. Ecological epigenetic studies are in their infancy, but it is paramount for the field to move forward to have detailed information about tissue and time dependence relationships in methylation to gain insights into if blood DNA methylation patterns can be a reliable bioindicator for changes in methylation that generate phenotypic variation in ecologically important traits.

5 Gene expression analysis and DNA methylation patterns of porcine somatic cell nuclear transfer blastocysts with high and low incidence of apoptosis

2019 ◽  
Vol 31 (1) ◽  
pp. 128
Author(s):  
L. Moley ◽  
R. Jones ◽  
R. Kaundal ◽  
A. Thomas ◽  
A. Benninghoff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Enrichment Analysis ◽  
Epigenetic Reprogramming ◽  
Reduced Representation ◽  
False Discovery ◽  
Reduced Representation Bisulfite Sequencing ◽  
Methylation Patterns ◽  
Dna Methylation Analysis ◽  
Go Terms

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n=13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P>0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced invivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.

scholarly journals epiGBS2: an improved protocol and automated snakemake workflow for highly multiplexed reduced representation bisulfite sequencing

2020 ◽  
Author(s):  
Fleur Gawehns ◽  
Maarten Postuma ◽  
Thomas P. van Gurp ◽  
Niels C. A. M. Wagemaker ◽  
Samar Fatma ◽  
...  
Keyword(s):  
Cytosine Methylation ◽  
Bisulfite Sequencing ◽  
De Novo ◽  
Bioinformatics Pipeline ◽  
Reduced Representation ◽  
Link Type ◽  
Time Investment ◽  
Reduced Representation Bisulfite Sequencing ◽  
Wide Range ◽  
Laboratory Protocol

AbstractepiGBS is an existing reduced representation bisulfite sequencing method to determine cytosine methylation and genetic polymorphisms de novo. Here, we present epiGBS2, an improved epiGBS laboratory protocol and user-friendly bioinformatics pipeline for a wide range of species with or without reference genome. epiGBS2 decreases costs and time investment and increases user-friendliness and reproducibility. The library protocol was adjusted to allow for a flexible choice of restriction enzymes and a double digest. Instead of fully methylated adapters, semi-methylated adapters are now used. The bioinformatics pipeline was improved in speed and integrated in the snakemake workflow management system, which now makes the pipeline easy to execute, modular, and parameter settings flexible. We also provide a detailed description of the laboratory protocol, an extensive manual of the bioinformatics pipeline, which is publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.3819996), and example output.

Abstract P175: Discovery And Validation Of Unique Associations Of DNA Methylation Regions With 24-hour Blood Pressure Phenotypes In African Americans

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Michelle L Roberts ◽  
Theodore A Kotchen ◽  
Xiaoqing Pan ◽  
Yingchuan Li ◽  
Chun Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
African Americans ◽  
Cumulative Effect ◽  
Epigenetic Mark ◽  
Discovery Cohort ◽  
Reduced Representation ◽  
Specific Pcr ◽  
Reduced Representation Bisulfite Sequencing ◽  
Sequencing Method ◽  
Independent Cohort

DNA methylation, an epigenetic mark, may reflect the interactions between DNA, environment, and lifestyle. It has been implicated in the development and progression of hypertension, a risk factor for cardiovascular disease. We hypothesize that regions of DNA methylation in blood cells can explain 24h BP phenotypes in African Americans. We performed Reduced Representation Bisulfite Sequencing (RRBS) in a discovery cohort of 281 African Americans. Several DNA methylation regions (MRs) were significantly associated with continuously monitored 24-h, daytime, or nighttime SBP, DBP, PP, and MAP after adjustments for covariates age, sex, and body mass index (False Discovery Rate (FDR) = 0.013 - 0.050). Each of these MRs explained a substantial portion of 24h BP variance, ranging from 6.5% - 9.4%. After FDR adjustment, there were no MRs significantly associated with clinic BPs (FDR > 0.1374), calculated by the average of 4 resting measurements (2 per arm) by sphygmomanometer. To interrogate specific regions of DNA methylation, our lab developed a potentially clinically applicable, deep, and targeted methylation sequencing method called Bisulfite-Specific PCR ULtrapLEx Targeted Sequencing (BULLET-Seq), and tested it in two reference samples for three MRs of interest. BULLET-Seq is able to accurately quantify the 10% changes in the dilution series when methylation rates ranged from ~40% to 90% (a chr19 methylation region; R 2 = 0.95 - 0.97) and can modestly measure these changes when rates range from ~2% to 4% (a chr5 region, R 2 = 0.82), and is questionable when methylation rates are below 2% (a chr13 region, R 2 = 0.03 - 0.27). Validation of the chr19 MR in an independent cohort (n=117) was performed in a single BULLET-Seq run. After covariate adjustment, the chr19 region was significantly associated with 24h BPs (SBP, DBP, and MAP; FDR < 0.05), confirming the findings from the discovery cohort. The MR accounted for up to 1.75% of BP variance in the 24h phenotypes. In conclusion, the reported DNA MRs have potential to be excellent markers for the cumulative effect of factors that influence 24h BPs and the BULLET-Seq workflow can be applied in clinical and population settings to screen up to thousands of patients.

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