This study was funded by NIH grant MH101392 (RSL) and support from the following awards and foundations: a NARSAD Young Investigator Award, Margaret Ann Price Investigator Fund, the James Wah Mood Disorders Scholar Fund via the Charles T. Bauer Foundation, Baker Foundation, and the Project Match Foundation (RSL).

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BSmooth:+from+whole+genome+bisulfite+sequencing+reads+to+differentially+methylated+regions.">BSmooth: from whole genome bisulfite sequencing reads to differentially methylated regions.</a> Genome Biology. 13 (10), R83 (2012).</li><li>Lee, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+corticosterone+exposure+increases+expression+and+decreases+deoxyribonucleic+acid+methylation+of+Fkbp5+in+mice.">Chronic corticosterone exposure increases expression and decreases deoxyribonucleic acid methylation of Fkbp5 in mice.</a> Endocrinology. 151 (9), 4332-4343 (2010).</li><li>Lee, R. 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The manuscript is part of a contest prize from Agilent Technologies.

In this study, we designed and implemented the Methyl-Seq platform for the rat genome. By demonstrating its utility with a rat model of stress, we demonstrated that the experimental and analytical pipeline can provide differentially methylated regions between two comparison groups.
To ensure a successful implementation of the platform, several critical steps need to be observed. First, initial DNA quality and quantity has a significant impact on the quality and quantity of the final Methyl-Seq library. We used a fluorometer, rather than a spectrophotometer, to ensure that our DNA measurement reflected the quantity of double-stranded DNA present. The bioanalyzer was used to measure the molecular size and quantity of DNA following shearing and after adapter ligation. Verifying the molecular size &#34;shift&#34; between these steps is crucial to confirm the presence of adapters at the ends of each DNA fragment that will undergo adapter-mediated PCR in the subsequent steps. The quantity of DNA remaining at the end of the adapter ligation step is also important, since at least 100 ng of the library product is needed at this step to ensure sufficient quantity is available after the target enrichment and bisulfite conversion steps. A final high-sensitivity measurement was performed on the constructed Methyl-Seq library so that the library can be properly diluted for subsequent clustering on the next-generation sequencer. Finally, bisulfite pyrosequencing was employed as a highly-quantitative, independent method to assess the accuracy of the analytical pipeline. The final validation using the original samples and replication using additional animals are crucial steps to ensure that the experiment can detect biologically significant changes in DNA methylation.
We also include several guidelines in the event of deviation from the protocol or if problems are encountered. First, it is possible to lose too much DNA during end-repair, adapter ligation, or magnetic bead purification steps. Alternatively, starting amounts of DNA could be small (&#60;200 ng) due to limited tissue/DNA availability or implementation of various enrichment methods such as fluorescence activated cell sorting. Increasing the cycle number during the two library amplification steps may be able to compensate for the excessive loss of DNA or low starting DNA amount throughout the library construction protocol. However, no more than an additional 2&#8211;3 cycles are recommended, as excessive template amplification is likely to lead to an increase in the number of duplicate reads being sequenced. These duplicates are excluded during the alignment step to prevent bias in percent methylation calculations. Second, if the average DNA size does not increase by more than 30 bps, check to ensure that the reagents are new, as T4 DNA polymerase, Klenow, and/or T4 ligase may be old. Commercially available replacement reagents can be used.
Additionally, it is possible that the predicted DMRs might not validate by pyrosequencing, where DNA methylation differences do not exist or are significantly less than those predicted by analysis. Poor validation of candidate regions is a problem too common for many genome-wide analyses, such as when pyrosequencing results do not confirm differential methylation or the effect size is much smaller than that predicted by the analysis. BSmooth is one analytical package that &#34;smoothes&#34; the methylation levels across a window of multiple CpGs. For the current experiment, BSmooth implicated a DMR whose methylation levels were validated by bisulfite pyrosequencing. However, there will likely be discrepancies between methylation levels predicted by BSmooth and those verified by pyrosequencing. The discrepancies arise from the smoothing function that estimates the average methylation values across all of the CpGs within a DMR, including consecutive CpGs that may differ in DNA methylation by more than 50% or CpGs whose methylation values were excluded due to sub-threshold read depth. R-packages such as MethylKit24 can be used to identify smaller windows of CpGs or even single CpGs whose methylation levels correlate strongly with those validated by pyrosequencing. Implementing different packages and testing their predicted regions or CpGs of differential methylation by pyrosequencing will ensure robustness of data. Alternatively, original Methyl-Seq libraries can be resequenced and added to the read files to increase read depth. Since determination of methylation levels are semi-quantitative and dictated by the number of reads [(# of CpGs)/(# of TpGs+CpGs)], increasing the read depth for a given CpG will increase the accuracy of its percent methylation value. In this study, we only considered CpGs whose methylation values were determined by at least ten reads and achieved an overall read coverage of 19x for each CpG.
The rat Methyl-Seq platform is not without its limitations. While it is more cost effective than whole-genome bisulfite sequencing, it is considerably more expensive than other methods. Nevertheless, most of the cost was for purchasing lanes on the sequencer and not for the capture system. Depending on the read depth necessary, with cross-tissue comparisons requiring less due to large (25&#8211;70%) differences12 in DNA methylation, the cost can be reduced by multiplexing more samples per lane and using a higher-capacity platform. Also, the sample preparation is more time-consuming than other methods. While similar to other pulldown approaches that incorporate next-generation sequencing, the added bisulfite conversion and purification steps add to the work load. Overall, the Methyl-Seq platform is a cost-effective alternative to whole-genome sequencing and provides base-pair resolution at more than 2.3 million CpGs, which is considerably more than those assayed by microarray-based platforms. To date, the commercially-available human and mouse Methyl-Seq platforms have been used to document alcohol-dependent changes in the macaque brain25,26, neurodevelopmental genes in the mouse brain9, and blood-brain targets of glucocorticoids10. Further, the ability to target specific regions regardless of sequence recognition by restriction enzymes makes it an ideal platform for cross-species comparisons. For this study, we designed the Methyl-Seq platform for the rat, for which many pharmacological, metabolic, and behavioral experiments are performed without the benefit of a genome-wide methylomic tool. Our data show that it can be used to detect DMRs in a rat model of stress and correlated other physiological parameters such as overall plasma CORT levels.
The Methyl-Seq platform is ideal for epigenetic experiments in animals with sequenced genomes that may not have enough experimental evidence documenting regulatory regions. When such regions are made available, additional regions may be custom-designed and attached to the current version. Further, the platform is ideal for comparative genomics, since the target enrichment is not constrained by restriction enzyme recognition. For instance, the promoter region of any gene of interest can be captured regardless of whether it harbors a specific restriction site. Similarly, any regulatory regions, such as those identified in mouse or humans, which are conserved in the genome of interest can be captured.

Advances in high-throughput sequencing have led to a wealth of genomic sequences for both model and non-model organisms. The availability of such sequences has facilitated research in genetics, comparative genomics, and transcriptomics. For instance, available genomic sequences are highly useful for aligning sequencing data from ChIP-Seq experiments that enrich DNA based on its association with histone modifications1, or bisulfite sequencing, which measures DNA methylation by detecting uracil formed from bisulfite conversion of unmethylated cytosines2. However, there have been delays in the implementation of epigenomic platforms that incorporate available genomic sequencing data in their design due to a lack of annotated data of species-specific regulatory sequences that can influence gene function. 
In particular, DNA methylation is one of the most widely studied epigenetic modifications on DNA that can leverage available genomic data for building a methylomic platform. One such example is an array-based platform for the human methylome3, which has been widely used in various disciplines from oncology to psychiatry4,5. Unfortunately, similar platforms for non-human animal models are scarce, as there are virtually no widely-used platforms that have taken advantage of the genomic sequence in their initial design.
A common method to assess the methylomic landscape of non-human animal models is reduced representation bisulfite sequencing (RRBS)6. This approach overcomes the cost of whole-genome bisulfite sequencing that, while providing a comprehensive methylomic landscape, provides lower read-depth coverage due to cost and limited functional information in large gene-poor areas of the genome2. RRBS involves restriction digest and size-selection of genomic DNA to enrich for highly GC-rich sequences such as CpG islands that are commonly found near gene promoters and thought to play a role in gene regulation7. While the RRBS method has been used in a number of important studies, its reliance on restriction enzymes is not without notable challenges and limitations. For instance, enrichment of GC-rich sequences in RRBS is entirely dependent on the presence of specific sequences recognized by the restriction enzyme and subsequent size selection by electrophoresis. This means that any genomic areas that do not contain these restriction sites are excluded during size selection. Also, cross-species comparisons are challenging unless the same restriction sites are present in the same loci among the different species. 
One approach to overcoming the limitations of RRBS is to use an enrichment method that takes advantage of the published genomic sequence in the design of the platform. The array-based human platform uses primer probes designed against specific CpGs for allele-specific (CG vs. TG after bisulfite conversion) target annealing and primer extension. Its design reflects not only the available human genomic sequence, but experimentally-verified regulatory regions acquired from multiple lines of inquiry, such as ENCODE and ENSEMBL8. Despite its wide use in human methylomic investigations, a similar platform does not exist for model animals. In addition, the array-based format places significant constraint on the surface area available for probe placement. In the past several years, efforts have been made to combine the target-specificity afforded by capture probe design and the high-throughput feature of next-generation sequencing. Such an endeavor has resulted in the sequencing-based target enrichment system for the mouse genome (mouse Methyl-Seq), which was used to identify brain-specific or glucocorticoid-induced differences in methylation9,10. Similar platforms for other model and non-model animals are needed to facilitate epigenomic research in these animals.
Here, we demonstrate the implementation of this novel platform to conduct methylomic analysis on the rat. The rat has served as an important animal model in pharmacology, metabolism, neuroendocrinology, and behavior. For example, there is an increasing need to understand the underlying mechanisms that give rise to drug toxicity, obesity, stress response, or drug addiction. A high-throughput platform capable of capturing methylomic changes associated with these conditions would increase our understanding of the mechanisms. Since the rat genome still lacks annotation for regulatory regions, we incorporated non-redundant promoters, CpG islands, island shores11, and previously identified GC-rich sequences into the rat Methyl-Seq platform12. 
To assess successful design and implementation of the SureSelect Target Enrichment (generically referred to as Methyl-Seq) platform for the rat genome, we employed a rat model of chronic variable stress (CVS)13 to identify differentially methylated regions between unstressed and stressed animals. Our platform design, protocol, and implementation may be useful for investigators who may want to conduct a comprehensive and unbiased epigenetic investigation on an organism whose genomic sequence is already available but remains poorly annotated.

<table><tbody><tr><td>Radioimmuno assay (RIA)</td><td>MP Biomedicals</td><td>7120126</td><td>Corticosterone, 125I labeled</td></tr><tr><td>Master Pure DNA Purification Kit</td><td>Epicentre/Illumina</td><td>MC85200</td><td></td></tr><tr><td>Thermal-LOK 2-Position Dry Heat Bath</td><td>USA Scientific</td><td>2510-1102</td><td>Used with 1.5 mL tubes</td></tr><tr><td>Vortex Genie 2</td><td>Fisher</td><td>12-812</td><td>Vortex Mixer</td></tr><tr><td>Ethyl alcohol, Pure</td><td>Sigma-Aldrich</td><td>E7023</td><td>100% Ethanol, molecular grade</td></tr><tr><td>Centrifuge 5424 R</td><td>Eppendorf</td><td>-</td><td>Must be capable of 20,000 x &lt;em&gt;g&lt;/em&gt;</td></tr><tr><td>Qubit 2.0</td><td>ThermoFisher Scientific</td><td>Q32866</td><td>Fluorometer</td></tr><tr><td>Qubit dsDNA BR Assay Kit</td><td>ThermoFisher Scientific</td><td>Q32850</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>ThermoFisher Scientific</td><td>Q32851</td><td>High sensitivity DNA detection reagents</td></tr><tr><td>Qubit Assay Tubes</td><td>ThermoFisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>SureSelectXT Rat Methyl-Seq Reagent Kit</td><td>Agilent Technologies</td><td>G9651A</td><td>Reagents for preparing the Methyl-Seq library</td></tr><tr><td>SureSelect Rat Methyl-Seq Capture Library</td><td>Agilent Technologies</td><td>931143</td><td>RNA baits for enrichment of rat targets</td></tr><tr><td>IDTE, pH 8.0</td><td>IDT DNA</td><td>11-05-01-09</td><td>10 mM TE, 0.1 mM EDTA</td></tr><tr><td>DNA LoBind Tube 1.5 mL</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>Covaris E-series or S-series</td><td>Covaris</td><td>-</td><td>Isothermal sonicator</td></tr><tr><td>microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm (25)</td><td>Covaris</td><td>520045</td><td></td></tr><tr><td>Water, Ultra Pure (Molecular Biology Grade)</td><td>Quality Biological</td><td>351-029-721</td><td></td></tr><tr><td>Veriti 96 Well-Thermal Cycler</td><td>Applied Biosystems</td><td>4375786</td><td></td></tr><tr><td>AMPure XP Beads</td><td>Beckman Coulter</td><td>A63880</td><td>DNA-Binding magnetic beads</td></tr><tr><td>96S Super Magnet</td><td>ALPAQUA</td><td>A001322</td><td>Magnetic plate for purification steps</td></tr><tr><td>2200 TapeStation</td><td>Agilent Technologies</td><td>G2965AA</td><td>Electrophresis-based bioanalyzer</td></tr><tr><td>D1000 ScreenTape</td><td>Agilent Technologies</td><td>5067-5582</td><td></td></tr><tr><td>D1000 ScreenTape High Sensitivity</td><td>Agilent Technologies</td><td>5067-5584</td><td></td></tr><tr><td>D1000 Reagents</td><td>Agilent Technologies</td><td>5067-5583</td><td></td></tr><tr><td>D1000 Reagents High Sensitivity</td><td>Agilent Technologies</td><td>5067-5585</td><td></td></tr><tr><td>DNA110 SpeedVac</td><td>ThermoFisher Scientific</td><td>-</td><td>Vacuum Concentrator</td></tr><tr><td>Dynabeads MyOne Streptavidin T1 magnetic beads</td><td>Invitrogen</td><td>65601</td><td>Streptavidin magnetic beads</td></tr><tr><td>Labquake Tube Rotator</td><td>ThermoFisher Scientific</td><td>415110Q</td><td>Nutator Mixer is also acceptable</td></tr><tr><td>EZ DNA Methylation-Gold Kit</td><td>Zymo Research</td><td>D5006</td><td>Bisulfite conversion kit. Contains Binding, Wash, Desulphonation, and Elution buffers</td></tr><tr><td>Illumina Hi-Seq 2500</td><td>Illumina</td><td>-</td><td>Next-generation sequencing machine</td></tr><tr><td>PCR and Pyrosequencing Primers</td><td>IDT DNA</td><td>Variable</td><td></td></tr><tr><td>Taq DNA Polymerase with ThermoPol Buffer - 2,000 units</td><td>New England BioLabs</td><td>M0267L</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Set</td><td>New England BioLabs</td><td>N0446S</td><td></td></tr><tr><td>Pyromark MD96</td><td>QIAGEN</td><td>-</td><td>Pyrosequencing machine</td></tr><tr><td>Ethyl Alcohol 200 Proof</td><td>Pharmco-Aaper</td><td>111000200</td><td>70% Ethanol solution</td></tr><tr><td>Sodium Hydroxide Pellets</td><td>Sigma-Aldrich</td><td>221465</td><td>0.2 M NaOH denature buffer solution</td></tr><tr><td>Tris (Base) from J.T. Baker</td><td>Fisher Scientific</td><td>02-004-508</td><td>10 mM Tris Acetate Buffer wash buffer solution</td></tr><tr><td>PyroMark Gold Q96 Reagents (50x96)</td><td>QIAGEN</td><td>972807</td><td>Reagents required for pyrosequencing</td></tr><tr><td>PyroMark Annealing Buffer</td><td>QIAGEN</td><td>979009</td><td></td></tr><tr><td>PyroMark Binding Buffer (200 mL)</td><td>QIAGEN</td><td>979006</td><td></td></tr><tr><td>Streptavidin Sepharose High Performance Beads</td><td>GE Healthcare</td><td>17-5113-01</td><td>Streptavidin-coated sepharose beads</td></tr><tr><td>PyroMark Q96 HS Plate</td><td>QIAGEN</td><td>979101</td><td>Pyrosequencing assay plate</td></tr><tr><td>Eppendorf Thermomixer R</td><td>Fisher Scientific</td><td>05-400-205</td><td>Plate mixer. 96-well block sold separately (cat. No 05-400-207)</td></tr><tr><td>SureDesign Website</td><td>Agilent Technologies</td><td>-</td><td>Target capture design software (https://earray.chem.agilent.com/suredesign/)</td></tr><tr><td>UCSC Genome Browser</td><td>University of California Santa Cruz</td><td>-</td><td>rat Nov 2004 rn4 assembly</td></tr><tr><td>Agilent Methyl-Seq Protocol</td><td>Agilent Technologies</td><td>-</td><td>https://www.agilent.com/cs/library/usermanuals/public/G7530-90002.pdf</td></tr></tbody></table>

a rat methyl-seq platform to identify epigenetic changes associated with stress exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Watch this Scientific Journal Video about A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure at JoVE.com

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

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10.7K Views.  Johns Hopkins School of Medicine. Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

Video: A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

Genetics

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Neuroscience

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid storage processes contributing to the development of the disease. We demonstrate a complete protocol from selection to epigenetic data analysis of patients with and without obesity. All steps from the protocol were tested and validated in a pilot study. 32 women participated in the study, in which 15 individuals were classified with obesity according to Body Mass Index (BMI) (45.1 &#177; 5.4 kg/m2); and 17 individuals were classified without obesity according to BMI (22.6 &#177; 1.8 kg/m2). In the group with obesity, 564 CpG sites related to fat mass were identified by linear regression analysis. The CpG sites were in the promoter regions. The differential analysis found 470 CpGs hypomethylated and 94 hypermethylated sites in individuals with obesity. The most hypomethylated enriched pathwayswere in the RUNX, WNT signaling, and response to hypoxia. The hypermethylated pathways were related to insulin secretion, glucagon signaling, and Ca2+. We conclude that the protocol effectively identified DNA methylation patterns and trait-related DNA methylation. These patterns could be associated with altered gene expression, affecting adipogenesis and lipid storage. Our results confirmed that an obesogenic lifestyle could promote epigenetic changes in human DNA.

The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps.

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Biology

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioengineering

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

Summary

Explore More Videos

Methyl-binding DNA capture Sequencing for Patient Tissues

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Methylated RNA Immunoprecipitation Assay to Study m⁵C Modification in Arabidopsis

Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

DNA Methylation: Bisulphite Modification and Analysis

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

Targeted DNA Methylation Analysis by Next-generation Sequencing