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    Epigenetics of Chronic Pain
    Epigenetics of Chronic Pain
    Epigenetics of Chronic Pain
    Ebook536 pages33 hours

    Epigenetics of Chronic Pain

    By Academic Press

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    About this ebook

    Epigenetics of Chronic Pain, Volume Nine, presents comprehensive information on the role of epigenetics in chronic pain sensitivity, providing a detailed, but accessible, view of the field from basic principles, to clinical application. Leading international researchers discuss essential mechanisms of chronic pain epigenetics, including the molecular processes of chromatin remodeling, histone modifications, and the microRNAs and noncoding RNAs involved in regulating genes tied to pain sensitivity. The influence of epigenetics in inflammatory, neuropathic, visceral and other pain models is examined, with data derived from epigenetic studies on peripheral and central mechanisms of pain sensitivity in animal models and clinical cases studies.

    The studies and case examples cited highlight therapeutic pathways of significance and next steps for researchers to develop epigenetic-based treatments for chronic pain. In recent years, epigenetic regulation of gene expression has been shown to play a central role in managing human pain sensitivity. Findings show that expression of many genes critical to increases or decreases in pain sensitivity are indeed regulated by DNA methylation and its enzymes, histone-involved chromatin remodeling, and noncoding RNAs, mainly microRNAs.

    • Compiles all known information on epigenetic regulation of chronic pain in one volume
    • Covers the basic functionality of epigenetic mechanisms involved in pain management, applications of recent research in understanding different types of chronic pain, and pathways for developing therapeutics
    • Leading international researchers from across academia, clinical settings, and the pharmaceutical industry discuss epigenetics in inflammatory, neuropathic, visceral, and other pain models in-depth
    • Enables clinicians, researchers, and pharmacologists to better understand and treat chronic pain
    LanguageEnglish
    PublisherAcademic Press
    Release dateOct 29, 2018
    ISBN9780128140710
    Epigenetics of Chronic Pain

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      Epigenetics of Chronic Pain - Academic Press

      Preface

      Allan Basbaum, Department of Anatomy, University California San Francisco, San Francisco, CA, United States

      A report from the Institute of Medicine found that almost 100 million Americans experience pain in a given year. Other studies indicate that 25 million of these individuals have pain that negatively affects their quality of life. These pain conditions can arise from traumatic injury, osteoarthritis, cancer, and even aging. Remarkably, pain persists long term in 15%–20% of elective surgery cases. Neuropathic pains, which result from damage to peripheral or CNS neurons, are particularly problematic, as even the best therapies are only effective in a small percentage of patients and the magnitude of pain relief provided by these interventions is small. The opioid epidemic in the United States highlights the need for new and safer approaches to managing chronic pain. Not surprisingly, the cost of managing these chronic pain conditions is enormous. In fact, estimates of the medical expenditure for managing chronic pain in the United States exceed $650,000,000 per year, an amount that is almost equal to the societal costs associated with treatment of cancer, diabetes, and heart disease combined.

      Recent studies that address the mechanisms that underlie the development of chronic pain after injury have focused on molecules and on the circuits in which they are engaged, from sensory neuron pathophysiology to altered CNS circuits. There is general agreement that the transition from acute pain, namely, the pain provoked by the original injury, to chronic pain results to a great extent, from a maladaptive plasticity of the nervous system. This maladaptive plasticity underlies the development of a profound hyperactivity of pain transmission circuits. The plasticity manifests at molecular, structural, biochemical, and physiological levels, with a major clinical feature being that normally innocuous stimuli are now painful (a condition called allodynia) and exaggerated pain occurs in response to normally painful stimuli (a condition called hyperalgesia). The persistent state of hypersensitivity involves remodeling and abnormal activity of injured axons, changes in synaptic strength at the level of the primary afferent, spinal cord, and brain, and perturbations in intracellular signaling pathways.

      Many of the pathophysiological contributors to chronic pain are also the product of long-lasting alterations in gene expression in neurons, at all levels of the pain transmission pathway. For this reason, factors that impact gene expression, in particular epigenetic influences, are now a focus of intense research. The epigenetic mechanisms include DNA methylation and demethylation, histone-regulated chromatin remodeling, and the contribution of microRNAs and other noncoding RNAs. Importantly, many studies have implicated epigenetic changes in a host of complex human diseases, including depression and addiction. The contribution of epigenetic regulation in neuronal processes is also highlighted by the dire neurodevelopmental consequences of mutations in the methyl-DNA binding protein MeCP2 and the de novo methyltransferase, DNMT3B, which, respectively, cause Rett syndrome and immunodeficiency, centromere instability, and facial anomalies syndrome (ICF).

      Until recently, however, there was less evidence for a comparable contribution of epigenetic changes with chronic pain, especially with a resolution that allows the assignment of epigenetic changes to specific genomic loci. For this reason, the publication of this new book on the epigenetics of pain is especially timely. The different chapters, in a very comprehensive manner, review the breadth of epigenetic mechanisms that regulate gene expression. The authors also discuss considerable preclinical and clinical evidence that there is, in fact, a significant contribution of epigenetic changes to the transition from acute to chronic pain.

      Different chapters dissect the epigenetic landscape of both acute and chronic pain, covering epigenetic contributions to inflammatory; neuropathic; and, importantly, even visceral pain. The latter is a major clinical problem that is often underrepresented in pain textbooks. The book will serve as a primer on epigenetics for the uninitiated, beginning with basic principles and then discussing recent research. Because of the breadth and depth of the coverage, the book will also be of particular interest to basic scientists and clinicians interested in possible epigenetic connections to the transition from acute to chronic pain. The ultimate objective of these studies is to obtain a better understanding of the molecular underpinnings of the maladaptive processes that can occur after injury. The traditional approach to therapy targets neurotransmitter receptors, transmembrane proteins (channels), and cytoplasmic cell signaling pathways, rather than the genetic elements that regulate expression of these different targets. Are epigenetic influences relevant? The wealth of studies described in this book suggests that the answer is an unequivocal yes, and not surprisingly, many potential therapeutic targets are introduced. Indeed, pharmacological and even viral-based genetics targeting of epigenetic mechanisms may provide a novel and potentially longer-lasting approach to pain management. Importantly, compounds that affect, with increasing specificity, epigenetic pathways are already entering the market. The possibility that these compounds will have utility in the management of chronic pain is an exciting one. This new text not only introduces readers to the epigenetic landscape of pain but also hopefully will encourage others to enter a growing research field, one with enormous potential benefit for patients.

      Chapter 1

      Epigenetic Tools in Chronic Pain Studies

      Guang Bai; Ke Ren    Department of Neural and Pain Sciences, University of Maryland Dental School, Baltimore, MD, United States

      Abstract

      Epigenetic regulation is involved in many biological and pathological processes. Initial studies demonstrate that epigenetic mechanisms play critical roles in the development of several preclinical models of hypersensitive nociception and a number of clinical chronic pain conditions. So far, a large number of various technologies have been developed to reveal molecular changes of epigenetic mechanisms and even modify epigenetic marks. To advance epigenetic studies of chronic pain, it is necessary to consider how to choose efficient approaches from these technological tools, appropriately analyze data collected, and translate it to clinical application.

      Keywords

      DNA methylation; DNA demethylation; Histone chromatin remodeling; Histone posttranslational modification; RNA modification; Noncoding RNA; MicroRNA; Next-generation sequencing; DNA bisulfite modification; Chromatin immunoprecipitation; HAT activator/inhibitor; HDAC inhibitor; Sirt activator/inhibitor; KMT inhibitor; PRMT inhibitor; KDM inhibitor; EpiEditor; CRISPR

      Introduction

      Since first proposed by Waddington (1942), the concept of epigenetics has been advanced on the bases of piling experimental data. Currently, most scientists agree that the epigenetics represents the regulation of gene expression mainly at transcription level in a way of DNA-sequence-independent manner, largely responding to environmental and intrinsic cues and possibly passing through individual or cell generation (Bonasio et al., 2010; Blancafort et al., 2013; Bai et al., 2015b, 2017; Nightingale, 2016). Molecular mechanisms involved in epigenetic regulation include at least DNA methylation, histone code-involved chromatin remodeling, and noncoding RNAs (ncRNA). The further layers of molecular mechanisms were proposed for RNA editing and DNA recoding (Mehler, 2008), as well as RNA posttranscriptional modifications (Roundtree and He, 2016; Saletore et al., 2012; Li et al., 2017). Enzymes catalyzing chemical modifications of DNA or histone are considered writer of epigenetic mark, enzymes removing modifications are eraser, and nuclear factors recognizing modifications are reader (Bai et al., 2015b; Lillico et al., 2016). Almost all of epigenetic marks can be analyzed both in a genome-wide-scale termed epigenome-wide association studies (EWAS) and in a gene-specific scale.

      Abnormal epigenetic regulation has been studied in many human diseases including various chronic pain conditions (Bai et al., 2015b, 2017). To further unwrap epigenetic mechanisms contributing to the pathological changes of chronic pain, appropriate tools are needed not only to assess changes of DNA methylation, chromatin remodeling, and ncRNA but also to evaluate the impacts of these changes on nociceptive behavior under physiological and pathological conditions. In this chapter, approaches to modify the level of these molecular mechanisms will be discussed in addition to summary of methods for molecular and biochemical measurements. In a broad sense, transgenic animals carrying modified epigenetic marks can serve as models for epigenetic studies of chronic pain. However, due to space limitation, this approach will not be discussed. In addition, although bioinformatic analysis is critical for EWAS, it is beyond the scope of this chapter and thus will not be discussed either.

      Measurement of Molecular Changes

      Various technologies have been developed to reveal or evaluate qualitative and quantitative changes of DNA methylation, chromatin remodeling, and ncRNA from single genes to EWAS studies. Many elegant reviews have already detailed these methodologies. Here, we want to provide readers a big picture regarding what appropriate approaches can be considered for studies of epigenetic mechanisms in chronic pain and will not detail methods, rather than citing these with protocols and providing our comments in general.

      Sample Preparation

      Pain or nociceptive signal is processed by a circuitry involving peripheral tissues (e.g., skin, muscle, viscera, and bones) and peripheral nervous system, as well as the central nervous system from the spinal cord to the brain stem, nuclei, and cortex (Tracey and Dickenson, 2012; Julius and Basbaum, 2001). Cells forming this circuitry include peripheral cells, neurons, and glial cells. Epigenetic changes in any of these cells may contribute to normal and abnormal processes of pain signal, such as a transition of acute pain to chronic pain following tissue damages (Ren and Dubner, 2016). In view of the location of epigenetic marks, subcellular structure, particularly nuclei, can be isolated, while neuron and glial cells can be separated by antibody-coupled flow cytometry from human and rodent brains (Guez-Barber et al., 2012; Feldmann et al., 2014; Matevossian and Akbarian, 2008). In addition, due to the selectivity of the blood-brain barrier permeability, different administration of reagents may access distinct part(s) of this circuitry. These points should be kept in mind when sampling tissues for biochemical and molecular analysis of epigenetic marks in chronic pain. In addition, recently, analysis of EWAS in single cells was introduced and is moving fast (Smallwood et al., 2014; Clark et al., 2016). Its sampling is more specifically relying on laser microdissection system from solid tissues such as the nervous system. In clinical studies, circulating cells are often the only available sample from patients suffering from various diseases such as chronic pain. Because it is well known that epigenetic regulation of DNA methylation, miRNA and other ncRNA expression, and even gene-specific chromatin remodeling characterize tissue specificity, changes of epigenetic marks in circulating cells or even body fluid always raise a question about how data generated from these samples are relevant to changes in the pain circuitry under chronic pain condition. In fact, collected data suggest that there is a kind of similarity between circulating cells and the nervous system in epigenetic changes (Bai et al., 2015b). It is well known that the quality of purified DNA and RNA largely impacts following assays, and thus, quality control should be performed routinely for chemical purity evaluated by absorbance at 260 and 280 nm of DNA and RNA and molecular integrity for DNA on low concentration of agarose electrophoresis and RNA on Agilent Bioanalyzer or both on Agilent TapeStation.

      DNA Methylation/Demethylation

      DNA methylation is a classic or prototype of epigenetic change showing methylation occurring on the 5′-position of the pyrimidine ring of cytosine residues in mammalian cells and considered a negative regulator in transcription regulation (Hendrich and Tweedie, 2003; Lan et al., 2010; Bird, 2002; Sweatt, 2009). Our understanding of DNA methylation and critical issues regarding the status of methylated DNA has been pointed out in our previous review (Bai et al., 2015b). To reinforce these concepts, several facts should be paid attention to when analyzing DNA methylation. First, methylated cytosine (5mC) occurs mostly in CpG dinucleotides located in repetitive sequences of the genome but much less in CpG island (CGI) in comparison with CGI shore and intergenic and intragenic loci. Second, most methylated CpGs are located in repetitive sequences of the genome on both or single alleles and on both strands (full) or one strand (hemimethylation) (Adams and Lindsay, 1993). Third, after the first report of non-CpG methylation occurring in the human PGC-1α promoter, methylation of cytosine followed by any nucleotide residue has been found universal in human or rodent genomes (Barrès et al., 2009; Bai et al., 2015b). Last, functional output may need methylation of a few CpGs, and methylated DNA may not necessarily suppress gene expression, while DNA methylation occurs in a broad range. For example, a report of differential methylated DNA studies in selected genes from three chromosomes of 12 different human tissues reveals that only one-third of methylated 5′ untranslated regions show inversely transcription reduction (Eckhardt et al., 2006). Studies of human tissues and cells revealed that 18,452 genomic regions close to 36% genes have intermediate methylation (Elliott et al., 2015). Another report of genome-wide studies of DNA methylation and mRNA profile in a human cell line indicated that exon 1 methylation is much more tightly linked to gene silencing than promoter methylation, while more downstream intragenic methylation is not associated with gene expression (Brenet et al., 2011). Taken together, functional confirmation with transcription change is necessary for DNA methylation readout.

      Technologies or approaches to evaluate methylated cytosine largely affect our understanding of DNA methylation. A couple of recent reviews well detailed the cons and pros of these technologies (Yong et al., 2016; Kurdyukov and Bullock, 2016; Couldrey and Cave, 2014). For example, data collected from next generation of DNA sequencing (NGS) after sodium bisulfite (BS) modification revealed that cytosine followed by any residue can be targeted by the DNA methylation machinery, particularly in the brain (Lister et al., 2013; Hovestadt et al., 2014; Kozlenkov et al., 2016). New technologies also helped verifying and modifying several concepts of DNA methylation that were proposed from earlier studies and should be considered when designing experiments, selecting approaches, and analyzing data, that is, specific pattern in cell and tissue, asymmetrical in doubled strands, heritable or transient, and tissue-specific (Razin and Riggs, 1980). Another new finding in recent years is the delineation of 5mC demethylation following the discovery of ten-eleven enzymes (TET 1–3) and the development of several new technologies. It has been known that 5mC is oxidized into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) catalyzed by TETs (Fig. 1). Thymine DNA glycosylase further converts 5fC and 5caC into unmodified cytosine (Ito et al., 2011; Spruijt et al., 2013; He et al., 2011; Rasmussen and Helin, 2016; Alaghband et al., 2016; Bochtler et al., 2017). All of these modified cytosines are epigenetic marks that can be recognized by epigenetic readers (Spruijt et al., 2013; Rasmussen and Helin, 2016).

      Fig. 1 Methylation and demethylation of cytosine in DNA.

      Experimental data suggest that 5mC demethylation occurs often for actively expressed genes such as those involved in memory in the brain (Miller et al., 2008; Guo et al., 2011; Li et al., 2013a). Critically, 5hmC shows the highest level in the brain supporting the notion that DNA demethylation is active in the nervous system and thus very likely involves processing pain signals (Wang et al., 2014). Initial works of altered expression of TET and the level of 5hmC in dorsal root ganglion neurons following peripheral nerve injury have been reported by two labs in different models recently, suggesting that DNA demethylation may participate in epigenetic mechanisms of chronic pain (Chamessian et al., 2017; Weng et al., 2017).

      Due to the nature of DNA methylation, historically, a large number of technologies have been developed and became the most complicated approaches. Many review articles and book series have been published aiming to introduce these technologies (Andersen and Tost, 2018; Beck, 2010; Kurdyukov and Bullock, 2016; Mills and Ramsahoye, 2002; Lizardi et al., 2017; Yong et al., 2016; Couldrey and Cave, 2014), many of which are commercialized (Kurdyukov and Bullock, 2016). Recommendations regarding how to select appropriate approach are also made by some reviews (Kurdyukov and Bullock, 2016; Yong et al., 2016; Couldrey and Cave, 2014). NIH Roadmap Epigenomics Project lists selected experimental protocols and data standards (www.roadmapepigenomics.org).

      Genome-wide association studies

      Methylation

      A number of methods were created to measure the total or global level of 5mC from isolated genomic DNA (gDNA). Some of these methods became commercially available. A more quantitative approach using mass spectrometry analysis was also reported although it requires more resources than most commercial kits (Fernandez et al., 2018; Tost and Gut, 2006). In addition, histochemically, the level of global 5mCs and even 5hmCs and 5caCs in individual cells can be detected by antibodies (Santos et al., 2002; Guo et al., 2014; Gu et al., 2011b). Although several antibodies become available for 5mC and each oxidized 5mCs, immunohistochemical studies are limited to embryos due to weak signals (Beaujean et al., 2018). However, the significance of global changes of 5mC level needs to be considered carefully since the majority of 5mCs are constant and located in repeats of heterochromosome (Bai et al., 2015b). Currently, the biological significance of these loci is unclear even though these sequences may be transcribed into ncRNA.

      Two types of technology play a central role in EWAS, microarray (chip or array) and so-called massively parallel DNA sequencing, deep sequencing or next generation of DNA sequencing (NGS that will be used in this review). Several methods linked to microarray were developed toward building up a DNA methylome. Early microarrays are mostly constructed using CGI and promoter regions either in clone or oligonucleotides (oligos). For example, Yan et al. described an CGI array (Yan et al., 2000, 2002), and University Health Network provides human and mouse CGI microarrays for academic users with very reasonable price (www.pmgenomics.ca/cpgmouse), while other human and mouse promoter arrays are available from Affymetrix, Agilent, and Illumina. However, all of these microarrays suffer from a number of limitations. First, they cannot differentiate methylated and unmethylated DNA directly and thus depend on prescreening or enrichment by means such as methylation-sensitive restriction digestion followed by adapter ligation and polymerase chain reaction (PCR) to eliminate unmethylated CGI (Schumacher et al., 2008; Yan et al., 2002), affinity purification with 5mC antibody (MeDIP) (Weber et al., 2005) or with methyl-CpG binding proteins (Zhang et al., 2006), and immunoprecipitation of methyl-CpG binding protein (Ballestar et al., 2003). Second, the standard of CGI varies with different calculations, and earlier microarrays utilized CGIs that were determined by high stringency and thus missed many (Bock et al., 2007). Third, 5mC outside CGIs occurs in high frequency and impact transcription. For example, only one-third of hypermethylated 5′ untranslated regions and promoters in selected three human chromosomes are reversely correlated with their transcription in tissue-specific expression (Eckhardt et al., 2006). Genomic tiling array may cover more ranges but cost similar in comparison with promoter/CGI arrays (Zhang et al., 2006; Lippman et al., 2005) (Affymetrix).

      The most commonly used microarray is the HumanMethylation450 BeadChip (HM450) that is derived from previous HM27 BeadChip and covers 485,764 potential 5mCs from CGIs, CGI shores, promoters, and some non-CpGs in the human genome (Bibikova et al., 2011). Each cytosine, mostly in CpGs, in BS-treated DNA is probed by two types of assay. Type I consists of two different probes specific for methylated and unmethylated Cs, correspondingly, which are detected by one color channel. Type II is constructed by one probe with degenerate site for methylated and unmethylated Cs detected with two colors after extension (Bibikova, 2016; Bibikova et al., 2011). In addition to being able to discriminate methylated CpG on both strands, HM450 also provides fast process, solid quantitative readout, and specific location of targets. However, HM450 contains a limited number of 5mCs in addition to the requirements of complicated bioinformatic analysis (Wu and Kuan, 2018; Morris and Beck, 2015). To expand CpG coverage, recently, Illumina launched human MethylationEPIC BeadChip (EPIC) that covers 853,307 potential 5mC sites including 333,265 sites from enhancer regions (Moran et al., 2016). This new version of BeadChip was already applied in studies of interstitial cystitis/bladder pain syndrome in women (Bradley et al., 2018). Interestingly, bioinformatic search revealed that HM450 and EPIC contain 13,715 and 19,420 probes, respectively, perfectly matching mouse genome after BS conversion, indicating potential application of BeadChips in mouse studies (Needhamsen et al., 2017; Wong et al., 2013). In recent years, application of microarray in DNA methylation analysis was fast being replaced by DNA sequencing technologies, particularly after enrichments for cost reduction (Andersen and Tost, 2018).

      DNA sequence can be determined following the extension of a well-designed DNA primer on homogenous molecule of purified plasmids, PCR amplicon, enzyme digest fragment, etc. by Sanger's or Maxima's method. Development of PCR, fluorescent probe tailed nucleotides, and capillary fractionation automated Sanger's method, which is currently available from many core facility and custom services inexpensively (Karger and Guttman, 2009). Since 2005, sequences of heterogeneous DNA fragments in small size in established libraries can be defined simultaneously by NGS (Margulies et al., 2005). Currently, a number of platforms for NGS have been used for DNA methylation analysis: Roche/454 based on pyrosequencing, ABI/SoLiD, and Illumina/SOLEXA (Lizardi et al., 2017). In general, to build up library, gDNA fragments are prepared by enzyme digestion or sonication and ligated to a predesigned adaptor that serves for PCR amplification and extension in massively parallel sequencing. Collected data are subject to bioinformatic analysis and illustration of all sequences in libraries prepared. More complicated samples need more intensively sequencing and bioinformatic analysis, thus resulting in higher cost. Protocols and reagents for NGS following chromatin immunoprecipitation (ChIP) have been compared for the best output (Sundaram et al., 2016). Detailed protocol is available (Sheaffer and Schug, 2016).

      It is a revolutionary step that BS treatment of DNA was found to convert unmethylated cytosine, but not 5mC, into thymidine in more than 99% efficiency (Leontiou et al., 2015), although minor error can be introduced by BS itself (as low as 0.09%) and following PCR (~ 0.2%) and its primer synthesis, as well as cloning bias in some products if applied (Warnecke et al., 2002; Genereux et al., 2008). It is recommended to check BS conversion rate (Holmes et al., 2014).

      Analysis of BS-modified gDNA using NGS reveals all survived cytosines as 5mCs regardless their position in CpG or CpH. This approach is termed the whole-genome BS sequencing (BS-seq) (Ziller et al., 2015; Taylor et al., 2007; Cokus et al., 2008; Daviaud et al., 2018; Li and Tollefsbol, 2011). Scientists at Applied Biosystems reported NGS analysis of methylated DNA in the whole human genome after figuring out how to treat DNA with BS (Ranade et al., 2009). Using the SOLiD system, they sequenced DNA library that was prepared from sonicated human gDNA fragments (60–90 base pair, bp) and treated by BS in the presence of denaturant formamide after ligated to adapters (Bormann Chung et al., 2010). This early protocol needs more than 2 μg starting gDNA, while current protocol handles 50–100 ng DNA (Daviaud et al., 2018) or even less than 10 ng DNA before BS (Edelmann and Scholten, 2018). To prevent BS-induced adapter damage and thus loss of DNA fragments, a post-BS adapter tagging (PBAT) was developed (Miura et al., 2012). For small amount of samples, BS-treated gDNAs can be amplified by whole-genome amplification methods (Mill et al., 2006). Certainly, less DNA represents less cell or genome number and thus may be less supportive for some scopes, dependent upon the working hypothesis.

      To obtain significant DNA methylation data, a concept of reduced representation bisulfite sequencing (RRBS) was introduced (Meissner et al., 2005). The initial RRBS protocol employed methylation-sensitive restriction enzyme digestion to enrich DNA fragments followed by a conventional adapter ligation-BS-PCR-cloning-seq strategy, and the sequencing was soon replaced by NGS (Meissner et al., 2005, 2008; Gu et al., 2011a). Since a large amount of expensive NGS cost is used for regions that do not show methylation alteration or not harbor genes nearby such as repetitive sequences (Bai et al., 2015b), the RRBS concept was quickly adopted and advanced with a number of alternative enrichments producing attractive data and significantly reducing the cost of NGS to less than 5% of what is for the entire genome (Xing et al., 2018; Li et al., 2015). For example, BS-treated DNA fragments were captured by DNA oligonucleotides (oligos) designed from CGIs and promoters followed by NGS as targeted BS-sequencing (TBS-seq) (Lee et al., 2011; Hodges et al., 2009; Deng et al., 2009; Ball et al., 2009; Nautiyal et al., 2010). Native methylated DNA fragments can be enriched for NGS by affinity binding of anti-5mC antibody (MeDIP-seq) (Li et al., 2018) combining with BS (BIsChIP-seq) (Statham et al., 2012), methylated DNA-binding proteins of MeCP2 (MethylCap-seq) (Brinkman et al., 2010), or MBD proteins (MIRA-seq) (Jung et al., 2015; Brenet et al., 2011). Unmethylated DNA fragments are enriched by biotinylation and capture of M.SssI-aminoalkylated cytosines for NGS (mTAG) (Kriukienė et al., 2013). On the basis of restriction enzyme sensitive to or dependent on methylation, both methylated and unmethylated DNA fragments are enriched by MRE-seq (Maunakea et al., 2010), Methyl-MAPS (Edwards et al., 2010), or HELP (Kriukienė et al., 2013). One weakness of RRBS or similar protocols is the potential bias during enrichment, missing some fragment artificially. To address this issue, Li et al. combined MeDIP-seq and MER-seq and significantly improved the coverage (Li et al., 2015).

      Recently, Yang and Scott took the advantage of single-molecule real-time (SMRT) sequencing (Pacific Biosciences) for long-range (10–15 kb) DNA sequencing and are able to directly analyze 5mCs within ~ 1.5 kb amplicon generated by an optimized BS-PCR (Yang and Scott, 2017). They also developed a software to process data collected. This approach bypasses subcloning each single molecule and is capable for reading PCR amplicons in multiplexed molecules and thus quantifying the changes of 5mCs in samples. Similar to direct sequencing of BS-PCR amplicons, 5mCs with low signal resulting from a low copy numbers in the sample may be missed even though they may represent critical cells in tissues having multiple cell types such as those from the nervous system.

      A new generation of DNA sequencing utilizes nanoparticles in processing samples without prelabeling and is able to differentiate 5mC from other bases (Feng et al., 2015). For example, Clarke and coworkers reported to use a protein (mutated α-hemolysin) nanopore with a covalently linked adapter and continuously read DNA sequence without any prelabeling with over 99% confidence (Clarke et al., 2009). Using the Oxford Nanopore Technologies MinION system, Simpson et al. read 5mC from CpG directly in a human cell line (Simpson et al., 2017). Chen et al. recently showed gold nanoparticles did the same job and also provided semiquantification of 5mC (Chen et al., 2016a). This approach sounds promising to process native or unmodified samples for methylated DNA, and their applications for an easy access and high-throughput format are expected.

      Methylome in single cells can be analyzed by single-cell GWBS (Zhu et al., 2018; Clark et al., 2016), and the details of protocols for single-cell BS-seq along with simple bioinformatic analysis are available (Clark et al., 2017; Edelmann and Scholten, 2018; Lee and Smallwood, 2018). A recently developed method termed single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) enriched open chromatin with DNMT and processed obtained DNA by BS-seq and RNA-seq (Clark et al., 2018). However, it is often actually multiple cells of the same kind that are analyzed in so-called single-cell technologies. A recent report claimed that gDNA from five halploid maize cells can be prepared into Illumina-compatible BS-seq library for NGS (Edelmann and Scholten, 2018). Nevertheless, this approach may be useful since epigenetic change in a few critical cells in the nervous system either peripheral or central may determine the fate of chronic pain.

      Demethylation

      Our understanding of DNA demethylation largely relies on technologies that differentiate oxidized cytosines (Booth et al., 2015; Peng et al., 2016). It is known that both 5hmC and 5mC are resistant to BS conversion into T. TET-assisted BS-seq (TAB-seq) protects native 5hmCs by glycosylation before TET conversion of all 5mCs into 5caCs and then identifies 5mCs revealed in BS-seq as 5hmCs along with the analysis of conventional BS-seq results (Yu et al., 2012, 2018). In oxidative BS-seq, 5hmCs are chemically converted to 5fCs to be read as T by subsequent BS-seq, and 5hmCs are identified in comparison with native BS-seq data (Booth et al., 2012). Reversely, in a reduced BS-seq, 5fCs are reduced back to 5hmCs that are read as C survived from BS conversion (Booth et al., 2014). In M.SssI methylase-assisted BS-seq, all nonmethylated Cs are methylated by the enzyme except 5fC and 5caC that are read as T in following BS-seq (Wu et al., 2014). To discriminate these two products, 5fCs can be detected after a hydroxylamine-based protection from BS modification (Song et al., 2013), while 5caCs are identified by an amine-based protection (Lu et al., 2013). Thereby, 5mC and its oxidized products can be fully differentiated if these protocols are used together (Qu et al., 2013). In addition to 5mC specific MeDIP-seq, antibody specific to 5hmC also applied in this technology to differentiate 5hmC (Zhao et al., 2014; Zhu et al., 2015). Since antibodies are available for 5fC and 5caC (Beaujean et al., 2018), we anticipate that application of these antibodies in MeDIP-seq may help enriching relevant DNA fragments for demethylation

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