Epigenetics Methods

Trygve O Tollefsbol

• Verlag: Academic Press
• Jahr: 2020
• ISBN:
  9780128194157
• Thema: Medicine
• Thema: Genetics in Medicine

In recent years, the field of epigenetics has grown significantly, driving new understanding of human developmental processes and disease expression, as well as advances in diagnostics and therapeutics. As the field of epigenetics continues to grow, methods and technologies have multiplied, resulting in a wide range of approaches and tools researchers might employ.

Epigenetics Methods offers comprehensive instruction in methods, protocols, and experimental approaches applied in field of epigenetics. Here, across thirty-five chapters, specialists offer step-by-step overviews of methods used to study various epigenetic mechanisms, as employed in basic and translational research. Leading the reader from fundamental to more advanced methods, the book begins with thorough instruction in DNA methylation techniques and gene or locus-specific methylation analyses, followed by histone modification methods, chromatin evaluation, enzyme analyses of histone methylation, and studies of non-coding RNAs as epigenetic modulators. Recently developed techniques and technologies discussed include single-cell epigenomics, epigenetic editing, computational epigenetics, systems biology epigenetic methods, and forensic epigenetic approaches. Epigenetics methods currently in-development, and their implication for future research, are also considered in-depth.

In addition, as with the wider life sciences, reproducibility across experiments, labs, and subdisciplines is a growing issue for epigenetics researchers. This volume provides consensus-driven methods instruction and overviews. Tollefsbol and contributing authors survey the range of existing methods; identify best practices, common themes, and challenges; and bring unity of approach to a diverse and ever-evolving field.

• Includes contributions by leading international investigators involved in epigenetic research and clinical and therapeutic application
• Integrates technology and translation with fundamental chapters on epigenetics methods, as well as chapters on more novel and advanced epigenetics methods
• Written at verbal and technical levels that can be understood by scientists and students alike
• Includes chapters on state-of-the-art techniques such as single-cell epigenomics, use of CRISPR/Cas9 for epigenetic editing, and epigenetics methods applied to forensics

Part One

Overview

Chapter One

Epigenetics methods in biological research

Trygve O. Tollefsbol^{a,b,c,d,e a} Department of Biology, University of Alabama at Birmingham, Birmingham, AL, United States
^b Center for Aging, University of Alabama at Birmingham, Birmingham, AL, United States
^c Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, United States
^d Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, AL, United States
^e Comprehensive Diabetes Center, University of Alabama at Birmingham, Birmingham, AL, United States

Abstract

Advances in epigenetics methods are critical to the field of epigenetics and have developed at a rapid rate over the past decade. These new methods have occurred not only at the gene- and locus-specific levels, but also encompass methods applied to the epigenome. Numerous novel methods have developed for elucidating the many mysteries of how DNA methylation and histone modifications control gene expression. Other molecular mechanisms such as those carried out by noncoding RNAs are also important to epigenetics and rapid advances in assessing noncoding RNAs have occurred as well. These new methods are being applied in ways that are completely changing conventional views of the role of epigenetics in health and disease.

Keywords:

Epigenetic; Methods; DNA methylation; Histone modification; Noncoding RNA

1 Introduction

Epigenetics has been a rapidly expanding field for quite some time and continues to develop at an exponential rate. A significant driving force in the expansion of this field has been the molecular tools that have been developed that allow researchers to study epigenetics in ways that were not previously possible. Many different epigenetic processes have been discovered and their application to biological processes have proven to be vast. This includes not only control of gene expression, but also specific processes such as X-chromosome inactivation and imprinting as well as broad biological processes including aging, cancer, autoimmunity and development [1–4]. In general, epigenetics refers to the cumulative heritable changes in phenotype that occur independent of the primary DNA sequence. The classic concept of epigenetics involved mitotic inheritance from cell to cell although a wealth of new information now supports epigenetic inheritance from generation to generation and is referred to as transgenerational epigenetic inheritance [5, 6].

DNA methylation has been the mainstay of classical epigenetics and consists of the enzymatic transfer of a methyl moiety from S-adenosylmethionine (SAM) to the 5-position of certain cytosines in CpG dinucleotides. The DNA methyltransferases (DNMTs) carry out DNA methylation and consist of three major types; DNMT1, DNMT3A and DNMT3B. The maintenance methyltransferase, DNMT1, is primarily responsible for preserving the methylation patterns with each cell replication while DNMT3A and DNMT3B carry out de novo methylation where a new methyl moiety is added at a CpG site that was not previously methylated.

Chromatin changes are also an important component of the epigenetic machinery and consist largely of posttranslational translational modifications of histones. These modifications are extensive and histone epigenetic marks often consist of acetylation or methylation that occur at specific amino acid residues in histones. Examples of histone-modifying enzymes are the histone acetyltransferases (HATs) and the histone deacetylases (HDACs) that add or remove acetyl groups from the histones, respectively. Both DNA methylation and histone modifications often collectively contribute to gene expression changes through molecular interactions that involve these major epigenetic processes [7].

In addition to DNA methylation and chromatin changes, noncoding RNAs are also an important and well-studied aspect of epigenetics. Noncoding RNAs are generally classified into two major groups, short or long forms, that can have a major impact on many biological processes such as gene expression. For example, microRNAs (miRNAs) can inhibit translation by binding to complementary mRNA leading to its degradation or the miRNA can inhibit transfer RNA by partially binding to the 3’ region of the mRNA and blocking the transfer RNA [8].

2 DNA methylation methods: Gene- or locus-specific methylation analyses

One of the most important breakthroughs in the area of epigenetics methods was the development of bisulfite methylation analyses. Bisulfite treatment of denatured DNA converts cytosine to uracil while 5-methylcytosine (5mC), the product of DNA methylation, is not converted. This principle led to numerous techniques that identify 5mC in DNA and methylation-specific PCR (MSP) is a frequent method that is used for evaluating DNA methylation changes in many different contexts. In fact, as described in Chapter 2 of this volume, MSP is often used for early diagnosis or prognosis of various diseases although it is not a highly sensitive technique unless it is monitored in real time in the quantitative MS-PCR (qMS-PCR) method. This latter modification of MS-PCR is precise and simple and has myriad applications to biological studies especially for biomarker screening purposes.

Another “gold-standard” technique that has significantly advanced DNA methylation analyses is pyrosequencing. Like qMS-PCR, it is also quantitative and can be performed with relative ease and can reveal regional DNA methylation changes or differences in DNA methylation at specific CpG dinucleotides (Chapter 3). More recently Droplet Digital PCR (ddPCR) has been developed and a modification of ddPCR, methylation-specific ddPCR (MS-ddPCR), has high sensitivity and precision for detecting DNA methylation. Chapter 4 describes how MS-ddPCR can be applied to detecting or quantifying rare methylated alleles in clinical samples which has been a challenge using more conventional approaches to the detection of cytosine methylation. This method, as is the case for qMS-PCR and pyrosequencing, has significant potential for applications to DNA methylation biomarker analyses.

3 DNA methylation methods: Global DNA methylation and methylomic analyses

Although gene- or locus-specific DNA methylation studies have contributed significantly to our knowledge of the role of DNA methylation in many biological processes, it is often advantageous to evaluate the overall changes in DNA methylation within cells, tissues or other samples. One of the global DNA methylation methods that is often used is the ELISA (enzyme-linked immunosorbent assay) technique that can detect not only 5mC but also 5-hydroxymethylcytosine (5hmC) that also often plays significant roles in epigenetic-based gene control. In this technique, DNA containing 5mC or 5hmC combines with anti-5mC or anti-5hmC and is compared to a fully methylated or hydroxymethylated control as reviewed in Chapter 5. The percentage difference reveals the global level of 5mC or 5hmC of the DNA that is being examined. This technique is most useful as a screening method for comparing the differences in global methylation between various samples. Another method for evaluating global DNA methylation is referred to as bisulfite PCR of repetitive genomic sequences. As covered in Chapter 6, various repetitive sequences in the genome such as Alu and LINE-1 repetitive elements undergo DNA methylation and due to their prevalence in the genome, their methylation status can indicate the overall methylation of genomic sequences. This global indicator of DNA methylation status has some challenges in methodology and interpretation. But it also has many applications especially with respect to biomarker analyses and applications to prognosis of disease.

The use of Illumina's bead technology for multiplexed analyses of CpG methylation in the human genome has been a popular approach to studies of DNA methylation. This technology, reviewed in Chapter 7, is based on the HumanMethylation (HM) BeadChip approach which allows profiling of the DNA methylation in human samples. This technique covers only less than 3% of the CpG sites in the human genome and is therefore not intended for complete methylome analyses. However, it can be highly useful when applied to population studies. Due in part to its limited coverage of CpG methylation, this is a relatively cost-effective technique that can be applied to large-scale studies of human samples such as is often the case in population-based studies.

There are many mapping approaches for global DNA methylation. One that has yielded a considerable amount of interest is reduced representation bisulfite sequencing (RRBS). In Chapter 8, Wan and Bell describe this technique that, like many other methods in epigenetics, is based on bisulfite conversion. It stands out, however, as a sensitive and cost-effective method that can be applied at a large scale but that also renders single-base resolution. CpG-dense regions are selected through restriction enzyme digestion of the genome and these regions are subjected to next-generational sequencing for profiling the methylome. Another technique, reviewed in Chapter 9, utilizes methylated DNA immunoprecipitation sequencing (MeDIP-seq) that provides information specifically about methylated regions. This method for DNA methylation analysis has provided a wealth of information over the past decade and is still a highly useful approach for determining DNA methylation changes when single-base resolution is a goal. This technique, which utilizes an antibody to 5mC for capturing fragmented genomic sequences followed by sequencing and differential methylation analyses, has the distinct advantage of relatively low cost for methylome studies.

Methylation sensitive restriction enzymes can be used to permit enrichment of hypo- or hypermethylated DNA segments that can then be evaluated using qPCR as covered in Chapter 10. The methylation restriction enzyme-based (MSRE) qPCR method facilitates analyses of minute amounts of DNA from plasma, for example, and can be a powerful tool for selecting optimal methylation markers for many different regions. In Chapter 11 Jessica Nordlund describes advances in whole genome methylomic sequencing which is generating considerable interest. Whole-genome bisulfite sequencing (WGSB) provides genome-wide profiling at single-base resolution. Expense has been a limiting factor in some cases although costs have been decreasing for this technique through short-read high throughput sequencing. This method often provides information about the methylation status of more than 85% of genomic cytosines at single-base resolution and is based on analyses of fragmented genomic DNA of short-read next-generation sequencing followed by ligation to adaptors, bisulfite treatment and creation of a sequencing library. Recently, a method has been developed based on BS-tagging that reduces potential redundant duplicate reads and that, along with other modifications, has significantly reduced the cost and increased the accuracy of WGBS. Overall, whole genome methylomic sequencing is showing considerable promise as a method for interrogating the methylome and it is expected that this technique will gather increasing use as additional modifications are developed that render this method more efficient and cost-effective.

4 DNA methylation methods: Additional technologies

Besides analyses of DNA methylation itself, it is often useful to also analyze the enzymatic machinery responsible for changes in DNA methylation. Many different assays have been developed for DNA methyltransferase activity measurements including those that are radioactivity-based, restriction enzyme-based, bisulfite conversion-based and affinity-based as covered in Chapter 12. Appropriate controls are important in these assays as well as use of optimal substrates since the DNMTs can vary greatly in their affinity for different types of methylation patterns in substrates.

In addition to 5mC changes in biological processes, variations in 5-hydroxymethylation (5hmC) are also important and have been suggested to play an important role in cancer development, embryonic stem cell pluripotency and cell fate transitions. In Chapter 13, Tomasz Jurkowski reviews many different approaches for the assessment of 5hmC with respect to its quantification and mapping. These methods are based on physicochemical analyses, restriction enzyme-based techniques, antibody-based affinity enrichment and other approaches to determine changes in 5hmC. Ultimately, analyses of 5hmC are projected to significantly increase as appreciation of the importance of the role of 5hmC in epigenetic processes also increase.

5 Histone modification methods

Chromatin immunoprecipitation (ChIP) assessment has become one of the most common tools for evaluating epigenetic processes and has greatly enhanced our knowledge of the important roles of chromatin in epigenetic control. ChIP combined with quantitative PCR (qPCR) or ChIP...

Inhaltsverzeichnis

• Cover image
• Title page
• Table of Contents
• Copyright
• Contributors
• Preface
• Part One: Overview
• Part Two: DNA methylation methods: Gene- or locus-specific methylation analyses
• Part Three: DNA methylation methods: Global DNA methylation and methylomic analyses
• Part Four: DNA methylation methods: Additional technologies
• Part Five: Histone modification methods
• Part Six: Epigenetic methods for evaluating chromatin higher order
• Part Seven: Enzyme analyses of histone methylation
• Part Eight: Assessment of non-coding RNA as an epigenetic modulator
• Part Nine: Additional epigenetics methods
• Part Ten: Future directions in epigenetics methodology
• Index