Discover millions of ebooks, audiobooks, and so much more with a free trial

Only $11.99/month after trial. Cancel anytime.

Epigenetic Technological Applications
Epigenetic Technological Applications
Epigenetic Technological Applications
Ebook1,117 pages67 hours

Epigenetic Technological Applications

Rating: 0 out of 5 stars

()

Read preview

About this ebook

Epigenetic Technological Applications is a compilation of state-of-the-art technologies involved in epigenetic research. Epigenetics is an exciting new field of biology research, and many technologies are invented and developed specifically for epigenetics study. With chapters covering the latest developments in crystallography, computational modeling, the uses of histones, and more, Epigenetic Technological Applications addresses the question of how these new ideas, procedures, and innovations can be applied to current epigenetics research, and how they can keep pushing discovery forward and beyond the epigenetic realm.

  • Discusses technologies that are critical for epigenetic research and application
  • Includes epigenetic applications for state-of-the-art technologies
  • Contains a global perspective on the future of epigenetics
LanguageEnglish
Release dateMay 30, 2015
ISBN9780128013274
Epigenetic Technological Applications

Related to Epigenetic Technological Applications

Related ebooks

Biology For You

View More

Related articles

Reviews for Epigenetic Technological Applications

Rating: 0 out of 5 stars
0 ratings

0 ratings0 reviews

What did you think?

Tap to rate

Review must be at least 10 words

    Book preview

    Epigenetic Technological Applications - Yujun George Zheng

    therapies.

    Chapter 1

    The State of the Art of Epigenetic Technologies

    Y. George Zheng,    Department of Pharmaceutical and Biochemical Sciences, The University of Georgia, Athens, Georgia, USA

    Epigenetics has become a focal point of biological research, significantly impacting biology, health, and medicine. Advances in technological developments enable great strides in epigenetic discovery. Numerous techniques have been developed not only to analyze epigenetic function, process and mechanism at the level of specific genes, but also to study epigenetic changes that occur on the genome-wide scale. The mapping of genome-wide DNA methylation can be readily achieved with ChIP-seq technology. Advances have also been made in technologies devised to assess the enzymes and binders that mediate epigenetic processes. Further, targeting various epigenetic regulators and processes with small molecule chemical probes represents an exciting therapeutic strategy for disease intervention. These technological advances collectively drive epigenetics research forward throughout the courses of discovery and invention, from basic biology to translational biomedical applications.

    Keywords

    Epigenetics; chromatin; histone; epigenetic technology; epigenetic therapy

    Chapter Outline

    1.1 Epigenetics and Chromatin Function 1

    1.2 Mechanisms of Epigenetic Regulation 2

    1.3 Technologies Are Critical for the Advancement of Epigenetic Discovery 8

    1.4 Epigenetic Inhibitors as Novel Therapeutics 11

    1.5 Perspective 12

    Acknowledgments 14

    References 14

    1.1 Epigenetics and Chromatin Function

    The field of epigenetics is rapidly booming, evolving, and expanding. The term epigenetics (the prefix epi comes from Greek, meaning over, upon, above, in addition to) was created by Waddington in the context of connecting developmental biology and genetics [1]. To date, epigenetics is most frequently referred to as the study of meiotically and mitotically heritable changes in gene activities without alterations in the genetic DNA sequence [1–4]. However, in recent years, it has been debated whether the heritability requirement of the term is necessary or too restrictive given that many (perhaps most) changes in gene activity modulated by chromatin modifications can occur in terminally differentiated and nondividing cells [3,5,6]. Examples include short-lived alterations in histone acetylation and methylation caused by DNA repair, cell-cycle phase, or transcription factor binding. Thus, there exist arguments in which epigenetic changes could be more broadly defined to encompass structural and biochemical alterations of the chromatin at any point in time under the condition that the genetic sequence is kept invariable [5,6]. Under this scheme, epigenetics does not merely refer to heritable chromatin states, but also embraces those that are transient or occur in nondividing cells. A new term, memigenetics, has recently been suggested to particularly denote transmissible epigenetics, which pertains to the propagation of a chromatin activity state across cell generations [5].

    Chromatin lies at the very core of epigenetic biology. In multicelled organisms, DNA, the macromolecule that contains genetic information, is packed into highly ordered chromatin complexes, accommodative to the narrow space of the nucleus. As the structural framework in which DNA embeds, chromatin executes various nuclear functions such as transcription, replication, and differentiation [7,8]. The basal structural building unit of the chromatin is the nucleosome, which is composed of a core histone octamer (containing two copies of each of H2A, H2B, H3, and H4 molecules) wrapped by 146 bp of DNA in approximately 1.7 turns [9,10]. The linker DNAs between neighboring nucleosomes connect nucleosome core particles, forming the 10-nm beads-on-a-string chromatin thread that can be observed under electron microscope [11,12]. A fifth histone, linker histone H1, binds to linker DNA and the entry/exit point of nucleosomal DNA, facilitating the folding of chromatin into higher order chromatin structures, such as the 30 nm filaments [13,14]. All five histones, H1, H2A, H2B, H3, and H4, are highly basic (the calculated pKa values are 10.3–11.4) and rich in positively charged amino acids, most of which are lysines and arginines. The strong basic physicochemical nature of the histones promotes their tight association with the DNA phosphate backbones to form stable chromatin complexes. The nucleosome complex structure represents a physical barrier that hampers the binding of gene regulatory factors, such as the RNA polymerases to gene promoters, thereby suppressing gene expression [15,16].

    1.2 Mechanisms of Epigenetic Regulation

    Though stable, the chromatin structure is a dynamic entity rather than static. Its structural state is directly associated with the activity status of the underlying DNA sequences. Regulation of chromatin structure and DNA activity is highly complex and involves several types of epigenetic control mechanisms, including DNA methylation, various histone posttranslational modifications (PTMs), exchange of canonical histones with histone variants, ATP-dependent chromatin remodeling, and recruitment of long and short noncoding RNAs (Figure 1.1). DNA 5-cytosine methylation is the most studied epigenetic mechanism in the field. Methylation of DNA occurs at the carbon-5 position of the cytosine ring, typically in a CpG dinucleotide context which is a characteristic feature of many eukaryotic genomes. Approximately 70% of CpG residues in the mammalian genomes are methylated, leaving a small portion of the genome methylation-free [17]. CpG methylation is an important mechanism for repressing the transcription of repeat elements and transposons, and plays a crucial role in genomic imprinting and X-chromosome inactivation [18].

    Figure 1.1 Chromatin structure and activity are epigenetically regulated by multiple molecular mechanisms, which include DNA modification on the cytosine residue with methyl and related one-carbon groups, assorted histone modifications, ATP-driven chromatin remodeling, exchange with histone variants, and interaction with noncoding RNAs.

    Compared to DNA methylation, histone modification encompasses much more diverse chemical groups, including methylation, acetylation, phosphorylation, citrullination, ubiquitination, SUMOylation, ADP-ribosylation, etc. [19]. So far, the majority of histone PTMs are found to be dynamically regulated. A huge number of histone modifiers that can enzymatically introduce or remove different histone marks in a site-specific manner (i.e. mark writers and erasers) have been discovered. Furthermore, epigenetic effectors (i.e. mark readers) can specifically recognize histone modifications, bringing about downstream outcomes. Acetylation is one of the most thoroughly studied histone modifications. The modification is dynamically controlled by the opposing enzyme activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Histone acetylation tags can be recognized by bromodomain-containing proteins, many of which are chromatin-associated regulators [20]. In general, histone acetylation is associated with open chromatin and transcriptional activation whereas histone deacetylation concurs to heterochromatin and gene silencing [21,22]. However, caution should be taken that this model may be overly simple because acetylated, open chromatin could also allow the access of repressor factors to the DNA template [23,24]. Further, HATs may repress gene activity via other mechanisms such as targeting transcriptional regulators [25,26].

    Histone methylation has more complicated patterns and has varied effects on gene activity which has no definite predictive index. Methylation can occur on the ω-amino group of arginine residues or the ε-amino group of lysine residues. Methylations of histone H3K4 and H3K36 are frequently associated with increasing transcriptional activity [27,28], whereas methylations of H3K9 and K3K27 are associated with gene silencing [29]. So far, multiple histone methyltransferases and demethylases were discovered which dynamically regulate the cellular methylation level. Reader proteins recognizing specific methylations have also been broadly studied. For example, chromodomain-containing proteins recognize methylated lysines and the tudor domain binds to methylated arginines [28,30].

    Although histone modifications were originally concentrated on the N-terminal tail region, current mass spectrometry (MS)-based detection reveals that they are widely spread over all the regions of the core histones, including the globular fold domain (Table 1.1) [31]. Many modifications are novel either because the modification sites are previously uncharted or the type of chemical groups is new. Based on a recent survey conducted by Huang et al. [31], 26% of amino acid residues in the four core histones were modified, and the majority of the modified sites are on arginine and lysine residues. It appears that virtually every lysine residue in the core histones is chemically modified. This raises many mechanistic questions regarding the timing, mechanism, and function of the modifications. For example, many residues in the globular domain are buried inside the nucleosome and cannot be accessed by enzymes. Posttranslational modifications of these residues must occur either prior to nucleosome assembly or following nucleosome disassembly. It is mysterious whether all histone modifications are enzymatically driven or whether some are due to accidental nonenzymatic chemical reactions. Further, it remains unknown whether all modifications have meaningful biological readouts or some of them are trivial and redundant. Illumination of biological consequences of individual histone modification tags will be a daunting challenge to epigeneticists.

    Table 1.1

    The Various Chromatin Modification Marks

    See a recent summary in [Ref. 31]

    1.3 Technologies are Critical for the Advancement of Epigenetic Discovery

    The progress in epigenetics is not possible without matching technologies. All the important breakthrough discoveries in epigenetic findings are made with the support of finely designed tools and strategies to detect, quantify, and image the chromatin state at multiple dimensions. A number of technologies were developed as the epigenetic field moves forward. These epigenetic technologies (Epi-Tech) include locus specific and genome-wide sequencing, creation of high-quality antibodies, chromatin functional assays, imaging tools, bioinformatics, and chemistry development.

    The field of DNA methylation has progressed considerably over the past decades. We now know that the 5-methyl group of cytosine can be enzymatically oxidized to hydroxymethyl, formyl, and even carboxyl groups [42]. Detecting and mapping technologies for studying DNA methylation and the newer cytosine modifications has gained rapid development [43]. In Chapters 2 and 3, colleagues from New England Biolabs and Emory University review the developing history of DNA methylation and discuss in detail various formats of high-throughput and high-resolution sequencing technologies for detecting cytosine modifications; the former emphasizes DNA methylation and hydroxymethylation, and the latter extends to cytosine 5-formylation and 5-carboxylation.

    Histone modifications are epigenetic tags as important as DNA methylation. The number and extent of histone modifications are far more complex than DNA methylation. MS has served as a powerful technology in epigenetics and has yielded significant impacts in providing a comprehensive picture of histone modifications [44] and understanding the consequence of the combinatorial histone modification patterns in the regulation of gene activities. Chapter 4 summarizes recent accomplishments of MS-based proteomics in the field of epigenetics, including histone sample preparation, enrichments of low abundant PTMs, different MS-based analytical strategies and bioinformatics tools for identification and quantification of histone PTMs, and histone variants. MS analysis is also able to determine chromatin modifications and interactome in a locus-specific manner with the combination of the chromatin immunoprecipitation (ChIP) technique.

    A number of technologies have been developed to study functions and dynamics of histone modification. Most of these technologies rely on antibodies that specifically recognize a particular histone modification mark. ChIP is a particularly useful common technique in studying epigenetic marks and has revolutionized chromatin research. ChIP in conjunction with DNA sequencing allows for assigning locus-specific functions to histone marks [45]. For example, ChIP analysis revealed that acetylation at H4 Lys16 is typically found in the promoters of transcriptionally active genes, whereas trimethylation at H3 Lys9 is observed predominantly in silenced heterochromatin [46]. Chapter 5 reviews the technique of ChIP and its application in elucidating histone modifications and chromatin regulators at specific loci. This technique is important in revealing that the different sequential recruitment of HAT and SWI/SNF-remodeling complex is promoter-type dependent: SWI/SNF-remodeling complex comes in first, followed by HATs on the HO gene promoter, while recruitment of HAT occurs prior to SWI/SNF on the IFN-β gene promoter [47,48].

    In addition to the four core histones, the linker histone H1 participates in the formation of higher order chromatin structures and adds another layer of complexity for the epigenetic regulation of gene expression. There are 11 mammalian H1 variants that are differentially regulated during development and cell cycle [49]. Chapter 6 discusses a suite of analytical tools and technologies used to study the functions of linker histone H1 variants, including qRT-PCR, HPLC analysis, gene targeting for H1 depletion, in vivo H1 tagging, ChIP of tagged H1 variants, and mutation analysis.

    Structural biology plays a critical role in elucidating the molecular basis of chromatin function and epigenetic regulation. As a powerful tool capable of providing atomic images of molecular recognition, macromolecular X-ray crystallography has greatly facilitated the mechanistic understanding of epigenetics phenomena in three-dimensional and atomic details. For instance, the 2.8 angstrom crystal structure of reconstituted nucleosomes resolved by Luger et al. in 1997 represents a real tour de force of discovery in the epigenetics field [10]. It provides atomic information regarding the nucleosome assembly and organization and has stimulated numerous efforts later to investigate the biochemistry and function of histone modifications in epigenetic regulation. Also, the rational drug design that is based on X-ray crystallographic and NMR structures of epigenetic proteins is an indispensable approach toward the development of novel selective epigenetic inhibitors. In Chapter 7, Zhao and Li review crystallographic studies of important epigenetic targets including writers, erasers, and readers of chromatin marks. As the authors point out, the application of macromolecular X-ray crystallography in epigenetic research will continue to provide novel mechanistic insights into epigenetic regulation and prompt the translation of mechanistic discoveries in epigenetics into therapeutic strategies impacting on medicine and its clinical ramifications.

    Histone proteins typically contain multiple PTM marks. The number is huge and even more complex is the possible vast combinations of histone marks. It remains challenging to elucidate the mechanistic role of individual marks as well as to identify the functional cross-talk between different PTMs. Molecular tools that enable dissecting function of individual histone marks and determining the biological consequence of cross-talks between different histone marks are highly demanded. In Chapter 8, Chatterjee and Dhall highlight chemical and genetic tools that allow access to homogeneously site-specifically modified histones for mechanistic investigations. Herein, there are two main strategies generally useful for preparing homogeneous histones containing desired PTM tags. The first strategy is direct biosynthesis via genetic codon expansion [50]. This method relies on an evolved orthogonal aminoacyl-tRNA synthetase/tRNA pair that specifically recognizes a specific unnatural amino acid (e.g. acetylated lysine) and directs its incorporation into recombinant proteins such as histones. The insertion site is precisely controlled by the position of an amber codon in the mRNA sequence of the protein target. An alternative approach is to use native chemical ligation and expressed protein ligation technology. Both methods have been extensively used in site-specific installation of histone PTM marks and yielded useful insights on the function of histone modifications [51]. In Chapter 9, Schutkowski and coworkers introduce the histone peptide microarray platform as a tool to profile epigenetic targets efficiently and comprehensively. In this technology, a histone microarray is made presenting thousands of histone peptides derived from human histone isoforms and also incorporating popular histone PTMs, such as lysine acylation, lysine/arginine methylation, serine/threonine/tyrosine phosphorylation, and various combinations of these modifications. This array approach enables rapid and efficient profiling of binding specificities of histone code readers and the determination of substrate specificity of the writers and erasers of histone marks. Importantly, addition of chemically introduced PTMs including recently discovered succinyl and malonyl lysines greatly facilitates to address the complex synergistic or antagonistic cross-talking relationship between different PTMs.

    Although many key PTMs, especially methylation and acetylation, were first studied on nucleosomal histones, of great interest is that they were later found to be present widely in nonhistone proteins. Hundreds of methylated proteins and thousands of acetylated proteins have been discovered from MS-based proteomic analysis. However, we do not understand well the dynamics of these modifications; especially the enzymatic modification mechanism is poorly understood [52]. In this regard, bioorthogonal chemical probes have been designed and applied to investigate protein substrates of HATs [53] and methyltransferases [54]. In Chapter 10, Luo reviews the techniques used for protein methylation profiling at the proteomic level. In particular, the author’s group developed a chemical biology strategy of bioorthogonal profiling of protein methylation, which is a powerful tool to dissect subsets of methylome for individual protein methyltransferases.

    High-throughput sequencing technologies have led to an explosion of biological data and provide the opportunity of mining disease markers and drug targets at the systematic level. Computational methods for epigenetic analysis have developed rapidly in answer to the call for efficient, accurate, fast management and processing of large volumes of data that are derived from epigenomic sequencing [43]. Bioinformatic and biostatistic tools are critical for dealing with large data sets and fishing out useful information in this omic era. In Chapter 11, Cheng et al. discuss the power of bioinformatic and biostatistic tools for the discovery of epigenetic biomarkers and drug targets in human diseases. Epigenetics is indispensable for the maintenance of cell phenotypes. The molecular mechanism for the inheritance of modification patterns over cell generations remains to be sorted out. In Chapter 12, Xing and coworkers illustrate a mathematical-modeling approach to explore the dynamics of histone modification, providing a theoretical understanding of the mechanisms of epigenetic inheritance.

    1.4 Epigenetic Inhibitors as Novel Therapeutics

    Epigenetic aberrations have been recognized in a diverse assortment of diseases such as cancer [55–58], obesity [59,60], diabetes [61,62], cardiovascular disorders [63,64], and neurological diseases [2,55,65,66]. Targeting epigenetic targets and processes represents an innovative strategy in disease treatment as it is aimed to restore the normal chromatin state of important genes, by either silencing or activating specific chromatin regulators. The success of DNMT and HDAC inhibitors as approved clinical drugs for treating hematological malignancies demonstrates that targeting epigenetic targets is a viable therapeutic approach [67]. Great efforts have been seen in recent years to develop chemical regulators of various epigenetic modifiers and readers [68–71].

    DNA methyltransferases (DNMTs) are promising epigenetic targets for the development of novel, single, or combined therapies in disease treatment [58,72]. 5-Azacytidine and decitabine are DNMT inhibitors approved by the Food and Drug Administration of the United States for the treatment of myelodysplastic syndromes (MDS). In Chapter 13, Medina-Franco et al. discuss the therapeutic significance of DNMTs in cancer and the current status of DNMT inhibitor development. The authors also present advances in experimental assays to screen compound collections, and elaborate on data sources and applications of chemoinformatics and molecular modeling that exemplify the significance of ChemEpinformatics in translational epigenetics.

    Dynamic regulation of cellular acetylation states is achieved by the functionally opposing enzymes – HATs and HDACs. Both HATs and HDACs are associated with various human diseases, particularly cancer. In Chapter 14, Luan et al. give a review on the current study of HAT enzymes, covering the biochemical assays for HAT activity measurement and the development of HAT chemical modulators. Development of small-molecule HDAC inhibitors was among the most advanced in epigenetic drug discovery [73]. Two HDAC inhibitors, romidepsin and SAHA, have already been used by cancer patients. In Chapter 15, Oyelere and coworkers review various assay technologies used for HDAC inhibitor screening and characterization, and highlight the strengths and weaknesses of each assay technology. It is worthwhile to stress that regulating HATs or HDACs is not the sole way of targeting the acetylation pathway. In recent years, success has also been witnessed in developing acetylation inhibitors by targeting the acetyl-lysine reader proteins, such as JQ1 and I-BET762 for the BET family of bromodomain proteins [74,75].

    Methylation is widespread in the nucleosomal histones and nonhistone proteins. In Chapter 16, Zeng and Xu comprehensively review various formats of histone methyltransferase assays including radiometric assay, fluorescence polarization assay, coupled enzyme assay, and antibody-based assay, with detailed discussion of their mechanisms, pros and cons, and potential applications. In Chapter 17, Li et al. give an account of inhibitor development for the family of histone methyltransferase enzymes, including lysine methyltransferases (PKMTs) and arginine methyltransferases (PRMTs).

    Lysine methylation is antagonized by lysine demethylases (KDMs). Jung and coworkers in Chapter 18 review the different types of histone demethylating enzymes and highlight key findings of their roles in the development of diseases. The authors present various formats of screening technologies for the discovery of histone demethylase inhibitors and demonstrated some key structural features of inhibitor binding. In Chapter 19, Whetstine et al. discuss several approaches for the purification and analysis of enzymatic activity of histone demethylases, and share technical protocols that allow for cellular characterization of these enzymes.

    Animal models are obligatory tools, along with cell culture–based systems, in the preclinical evaluation of epigenetic inhibitors. Promising drugs from biochemical or cellular screening usually proceed to be tested in animal models which provide data on dosing, toxicity, and other important pharmacological attributes that prelude to safe and efficient clinical trials. In Chapter 20, Huang et al. describe the use of animal models (both GEMMs and xenotransplant) for the evaluation and development of epigenetic inhibitors. The advantages, challenges, and limitations associated with animal models are discussed.

    1.5 Perspective

    Epigenetic research is rapidly flourishing and many directions are emerging. With many new sites and new types of modifications discovered in histones, precise yet systematic studies are warranted to assess the functions of those modifications. Also, proteomics studies have shown that most modifications found on histones occur across a myriad of proteins in and out of the nucleus, suggesting regulatory roles of these PTMs in cellular homeostasis. Thus, chromatin modification is not the sole domain of histone modifiers and readers. Dissecting the dynamics and functions of the PTM marks that were originally uncovered in chromosomal histones in a much larger context, across multiple cellular organelles and in different animal models, will provide a better, more precise understanding of their biological functions. Elucidating the broad substrate specificity and non-chromatin function of epigenetic proteins is expected. These efforts will in turn benefit the understanding of their regulatory function in chromatin restructuring and epigenetic inheritance. It could be speculated that histone modification is the result of a sophisticated selection of the bulk pool of posttranslational modification biochemistry during the history of evolution, which eventually manifests itself with specialized function for stabilization and regulation of the genetic material.

    The connection of epigenetics with metabolism is attracting intensive attention. Methyl donor nutrients (e.g. folate, glycine, and betaine) can increase DNA methylation, thereby altering gene expression [76]. Friis et al. [77] showed that inducing glucose catabolism and production of acetyl-CoA in quiescent cells activates global histone acetylation. Illumination of the functional linkage of metabolism to epigenetics is important, not only for inventing new disease therapy, but also for providing cognitive guidelines for disease prevention and long-term health maintenance. We know that there are many environmental mutagens that can cause cancer-related genetic mutations. Analogously, a number of efforts have been made to investigate the mechanism of environmental chemicals in acting as epimutagens to affect the epigenome of somatic cells and reproductive cells, which may lead to pathophenotypes at later stages [78,79]. The influence of nutrition on epigenetic states belongs to the subfield of nutri-epigenomics [80]. Possibly, many natural products, dietary supplements, and food components are essential to maintain and propagate the healthy state of the epigenotype. Alteration of nutritional compositions may lead to chronic accumulation of unstable epigenetic marks, which cumulatively promote the predisposition to tumorigenesis or other pathological transformations. Certainly, these new epigenetic findings will provide invaluable information for recommending healthy diet styles for early disease prevention.

    Given the great significance of epigenetic inhibitors in the pharmaceutical industry, development of drug-like inhibitors for various epigenetic targets will continue to be an important research focus in this field. Integration of in silico design and high-throughput screening of chemical libraries is an effective approach to discovering new drug leads. Natural compounds represent a rich source of chemical structures that could be used for the design of epigenetic drugs [81]. In pursuit of epi-drugs, discrimination of the selected target as a disease marker vs. a therapeutic target will be a priority step. One would wish to target only important driver genes (those that are epigenetically indispensable for disease to occur) instead of passenger genes (those that are epigenetically altered but are dispensable for disease to occur) [58]. Investigating mechanisms of drug action in vivo is critical. This is important for at least two reasons: first, many histone-modifying enzymes have numerous non-chromatin targets. For instance, thousands of cellular proteins were found to contain acetylated lysines, highlighting that histones are just the tip of the iceberg of the entire protein acetylome. Under this scenario, pharmacologic impacts of those inhibitors that target HATs or HDACs could be caused by blockage of acetylation-regulated pathways that are not associated with epigenetics. Another reason is regarding the polypharmacologic properties of small molecule drugs. Most drugs have multiple targets in the cell, so it is critical to examine whether the pharmacologic effect caused by the drug is authentically due to its action on epigenetic targets.

    In keeping up the pace with the advancement of epigenetic science, Epi-Tech will continue to play indispensable roles in the epigenetic field. The invention of powerful techniques to interrogate function and mechanism of the epigenome and epigenetic regulators has driven the remarkable advances in epigenetics. Numerous methods have been developed that provide means to accurately assess epigenetic processes, most notably DNA methylation and chromatin modifications as the most prevalent epigenetic mechanisms. The technical methods described in this book represent some of the exciting developments in this area. Advances in epigenetics are occurring at a remarkable pace and it is no doubt that this area will continue to herald critical breakthroughs in years to come. We anticipate that the advances in Epi-Tech will continually illuminate unresolved problems and further advance the field of epigenetics. To determine the changes of the epigenome under various contexts and environmental conditions, sequencing DNA methylome with improved technologies, such as SeqCapEpi, will likely become more financially inexpensive and accessible to investigators [82]. Deep sequencing coupled with Hi-C technology is a powerful approach for mapping genetic and epigenetic states with three-dimensional chromatin conformational structures [83,84]. The combination of microfluidic technology, modification-specific antibodies, and single-molecule imaging techniques enables measuring the degree of co-localization of histone modifications and DNA methylation on single nucleosomes [85,86]. The single molecule approach likely will play a more important role in chromatin analysis [87]. One advantage of the single-molecule method is its overcoming of cell source limitation. This is important for many cases; for example, progenitor cells from animals are often limited, which does not allow for standard ChIP analysis. Along with the rapid advance in epigenomic mapping using sophisticated technologies have come increasing challenges in managing massive amounts of information. Further developments in computational epigenetics is compulsory in deciphering the colossal amounts of data derived from experimental epigenomics [43]. With new progress, many key questions in this area, such as epigenetic etiology of disease, epigenetic inheritance, impact by nature and nurture, connection with mental health, and predictable pharmaco-epigenomics, will become illuminated in greater width and depth.

    Acknowledgments

    I acknowledge the National Institutes of Health and the American Heart Association for financial support. I thank H. Hu and K. Qian for providing the figure and the table in this chapter.

    References

    1. Holliday R. Epigenetics: a historical overview. Epigenetics. 2006;1(2):76–80.

    2. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429(6990):457–463.

    3. Felsenfeld G. The evolution of epigenetics. Perspect Biol Med. 2014;57(1):132–148.

    4. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457–463.

    5. Mann JR. Epigenetics and memigenetics. Cell Mol Life Sci. 2014;71(7):1117–1122.

    6. Bird A. Perceptions of epigenetics. Nature. 2007;447(7143):396–398.

    7. Ravindra KC, Narayan V, Lushington GH, Peterson BR, Prabhu KS. Targeting of histone acetyltransferase p300 by cyclopentenone prostaglandin Delta(12)-PGJ(2) through covalent binding to Cys(1438). Chem Res Toxicol. 2012;25(2):337–347.

    8. Campos EI, Reinberg D. Histones: annotating chromatin. Annu Rev Genet. 2009;43:559–599.

    9. Davis PKB, Brackman RK. Chromatic remodeling and cancer. Cancer Biol Ther. 2003;2(1):22–29.

    10. Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ. Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature. 1997;389(6648):251–260.

    11. Olins AL, Olins DE. Spheroid chromatin units (v bodies). Science. 1974;183(4122):330–332.

    12. Woodcock CL, Safer JP, Stanchfield JE. Structural repeating units in chromatin I Evidence for their general occurrence. Exp Cell Res. 1976;97:101–110.

    13. van Holde KE. Chromatin (Springer Series in Molecular and Cell Biology) 1988 ed. Springer 1988; p. 497.

    14. Wolffe A. Chromatin: Structure and function San Diego, CA: Elsevier Science & Technology Books; 1999.

    15. Lenfant F, Mann RK, Thomsen B, Ling X, Grunstein M. All four core histone N-termini contain sequences required for the repression of basal transcription in yeast. Embo J. 1996;15(15):3974–3985.

    16. Gregory PD, Wagner K, Horz W. Histone acetylation and chromatin remodeling. Exp Cell Res. 2001;265(2):195–202.

    17. Zheng YG, Wu J, Chen Z, Goodman M. Chemical regulation of epigenetic modifications: opportunities for new cancer therapy. Med Res Rev. 2008;28(5):645–687.

    18. Reik W. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature. 2007;447(7143):425–432.

    19. Buczek-Thomas JA, Hsia E, Rich CB, Foster JA, Nugent MA. Inhibition of histone acetyltransferase by glycosaminoglycans. J Cell Biochem. 2008;105(1):108–120.

    20. Marmorstein R, Zhou MM. Writers and readers of histone acetylation: structure, mechanism, and inhibition. Cold Spring Harb Perspect Biol. 2014;6(7):a018762.

    21. Kouzarides T. Chromatin modifications and their function. Cell. 2007;128:693–705.

    22. Grunstein M. Histone acetylation in chromatin structure and transcription. Nature. 1997;389(6649):349–352.

    23. Ooi L, Belyaev ND, Miyake K, Wood IC, Buckley NJ. BRG1 chromatin remodeling activity is required for efficient chromatin binding by repressor element 1-silencing transcription factor (REST) and facilitates REST-mediated repression. J Biol Chem. 2006;281(51):38974–38980.

    24. Wu SY, Chiang CM. The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation. J Biol Chem. 2007;282(18):13141–13145.

    25. Eckey M, Kraft F, Kob R, et al. The corepressor activity of Alien is controlled by CREB-binding protein/p300. Febs J. 2013;280(8):1861–1868.

    26. Perez-Campo FM, Costa G, Lie ALM, Stifani S, Kouskoff V, Lacaud G. MOZ-mediated repression of p16(INK) (4) (a) is critical for the self-renewal of neural and hematopoietic stem cells. Stem Cells. 2014;32(6):1591–1601.

    27. Wagner EJ, Carpenter PB. Understanding the language of Lys36 methylation at histone H3. Nat Rev Mol Cell Biol. 2012;13(2):115–126.

    28. Ruthenburg AJ, Li H, Patel DJ, Allis CD. Multivalent engagement of chromatin modifications by linked binding modules. Nat Rev. 2007;8(12):983–994.

    29. Barski A, Cuddapah S, Cui K, et al. High-resolution profiling of histone methylations in the human genome. Cell. 2007;129(4):823–837.

    30. Taverna SD, Li H, Ruthenburg AJ, Allis CD, Patel DJ. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol. 2007;14(11):1025–1040.

    31. Huang H, Sabari BR, Garcia BA, Allis CD, Zhao Y. SnapShot: histone modifications. Cell. 2014;159 458–458.e1.

    32. Sarg B, Lopez R, Lindner H, Ponte I, Suau P, Roque A. Identification of novel post-translational modifications in linker histones from chicken erythrocytes. J Proteomics. 2015;113:162–177.

    33. Wilson JP, Raghavan AS, Yang YY, Charron G, Hang HC. Proteomic analysis of fatty-acylated proteins in mammalian cells with chemical reporters reveals S-acylation of histone H3 variants. Mol Cell Proteomics. 2011;10 M110 001198.

    34. Unoki M, Masuda A, Dohmae N, et al. Lysyl 5-hydroxylation, a novel histone modification, by Jumonji domain containing 6 (JMJD6). J Biol Chem. 2013;288(9):6053–6062.

    35. Nathan D, Ingvarsdottir K, Sterner DE, et al. Histone sumoylation is a negative regulator in Saccharomyces cerevisiae and shows dynamic interplay with positive-acting histone modifications. Genes Dev. 2006;20(8):966–976.

    36. Pestinger V, Wijeratne SS, Rodriguez-Melendez R, Zempleni J. Novel histone biotinylation marks are enriched in repeat regions and participate in repression of transcriptionally competent genes. J Nutr Biochem. 2011;22(4):328–333.

    37. Hassan YI, Zempleni J. A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase. Nutr Rev. 2008;66(12):721–725.

    38. Nelson CJ, Santos-Rosa H, Kouzarides T. Proline isomerization of histone H3 regulates lysine methylation and gene expression. Cell. 2006;126(5):905–916.

    39. Howe FS, Boubriak I, Sale MJ, et al. Lysine acetylation controls local protein conformation by influencing proline isomerization. Mol Cell. 2014;55(5):733–744.

    40. Raghuram N, Strickfaden H, McDonald D, et al. Pin1 promotes histone H1 dephosphorylation and stabilizes its binding to chromatin. J Cell Biol. 2013;203(1):57–71.

    41. Hanes SD. Prolyl isomerases in gene transcription. Biochim Biophys Acta 2014.

    42. Xu Z, Li H, Jin P. Epigenetics-based therapeutics for neurodegenerative disorders. Curr Transl Geriatr Exp Gerontol Rep. 2012;1(4):229–236.

    43. Tollefsbol TO. Advances in epigenetic technology. Methods Mol Biol. 2011;791:1–10.

    44. Garcia BA, Pesavento JJ, Mizzen CA, Kelleher NL. Pervasive combinatorial modification of histone H3 in human cells. Nat Methods. 2007;4(6):487–489.

    45. Schmidt D, Wilson MD, Spyrou C, Brown GD, Hadfield J, Odom DT. ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods. 2009;48(3):240–248.

    46. Wang Z, Zang C, Rosenfeld JA, et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet. 2008;40(7):897–903.

    47. Krebs JE, Kuo MH, Allis CD, Peterson CL. Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. Genes Dev. 1999;13(11):1412–1421.

    48. Agalioti T, Chen G, Thanos D. Deciphering the transcriptional histone acetylation code for a human gene. Cell. 2002;111(3):381–392.

    49. Happel N, Doenecke D. Histone H1 and its isoforms: contribution to chromatin structure and function. Gene. 2009;431(1–2):1–12.

    50. Liu CC, Schultz PG. Adding new chemistries to the genetic code. Annu Rev Biochem. 2010;79:413–444.

    51. Li KK, Luo C, Wang D, Jiang H, Zheng YG. Chemical and biochemical approaches in the study of histone methylation and demethylation. Med Res Rev. 2012;32(4):815–867.

    52. Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol. 2014;15(8):536–550.

    53. Yang C, Mi J, Feng Y, et al. Labeling lysine acetyltransferase substrates with engineered enzymes and functionalized cofactor surrogates. J Am Chem Soc. 2013;135(21):7791–7794.

    54. Islam K, Bothwell I, Chen Y, et al. Bioorthogonal profiling of protein methylation using azido derivative of S-adenosyl-L-methionine. J Am Chem Soc. 2012;134(13):5909–5915.

    55. Fouse SD, Costello JF. Epigenetics of neurological cancers. Future Oncol. 2009;5(10):1615–1629.

    56. Gnyszka A, Jastrzebski Z, Flis S. DNA methyltransferase inhibitors and their emerging role in epigenetic therapy of cancer. Anticancer Res. 2013;33(8):2989–2996.

    57. Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy. Cell. 2012;150(1):12–27.

    58. Kelly TK, De Carvalho DD, Jones PA. Epigenetic modifications as therapeutic targets. Nat Biotechnol. 2010;28(10):1069–1078.

    59. Iyer A, Fairlie DP, Brown L. Lysine acetylation in obesity, diabetes and metabolic disease. Immunol Cell Biol. 2012;90(1):39–46.

    60. Kendrick AA, Choudhury M, Rahman SM, et al. Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation. Biochem J. 2011;433(3):505–514.

    61. Villeneuve LM, Natarajan R. The role of epigenetics in the pathology of diabetic complications. Am J Physiol Renal Physiol. 2010;299(1):F14–F25.

    62. Cooper ME, El-Osta A. Epigenetics: mechanisms and implications for diabetic complications. Circ Res. 2010;107(12):1403–1413.

    63. McKinsey TA, Olson EN. Cardiac histone acetylation – therapeutic opportunities abound. Trends Genet. 2004;20(4):206–213.

    64. Abi Khalil C. The emerging role of epigenetics in cardiovascular disease. Ther Adv Chronic Dis. 2014;5(4):178–187.

    65. Feng J, Fan G. The role of DNA methylation in the central nervous system and neuropsychiatric disorders. Int Rev Neurobiol. 2009;89:67–84.

    66. Valor LM, Viosca J, Lopez-Atalaya JP, Barco A. Lysine acetyltransferases CBP and p300 as therapeutic targets in cognitive and neurodegenerative disorders. Curr Pharm Des. 2013;19(28):5051–5064.

    67. Minucci S, Pelicci PG. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer. 2006;6(1):38–51.

    68. Miyamoto K, Ushijima T. Diagnostic and therapeutic applications of epigenetics. Jpn J Clin Oncol. 2005;35(6):293–301.

    69. Zelent A, Waxman S, Carducci M, Wright J, Zweibel J, Gore SD. State of the translational science: summary of Baltimore workshop on gene re-expression as a therapeutic target in cancer January 2003. Clin Cancer Res. 2004;10(14):4622–4629.

    70. Gilbert J, Gore SD, Herman JG, Carducci MA. The clinical application of targeting cancer through histone acetylation and hypomethylation. Clin Cancer Res. 2004;10(14):4589–4596.

    71. Mai A, Altucci L. Epi-drugs to fight cancer: from chemistry to cancer treatment, the road ahead. Int J Biochem Cell Biol. 2009;41(1):199–213.

    72. Gros C, Fahy J, Halby L, et al. DNA methylation inhibitors in cancer: recent and future approaches. Biochimie. 2012;94(11):2280–2296.

    73. Gryder BE, Sodji QH, Oyelere AK. Targeted cancer therapy: giving histone deacetylase inhibitors all they need to succeed. Future Med Chem. 2012;4(4):505–524.

    74. Filippakopoulos P, Qi J, Picaud S, et al. Selective inhibition of BET bromodomains. Nature. 2010;468(7327):1067–1073.

    75. Mirguet O, Gosmini R, Toum J, et al. Discovery of epigenetic regulator I-BET762: lead optimization to afford a clinical candidate inhibitor of the BET bromodomains. J Med Chem. 2013;56(19):7501–7515.

    76. Handy DE, Castro R, Loscalzo J. Epigenetic modifications: basic mechanisms and role in cardiovascular disease. Circulation. 2011;123(19):2145–2156.

    77. Friis RM, Wu BP, Reinke SN, Hockman DJ, Sykes BD, Schultz MC. A glycolytic burst drives glucose induction of global histone acetylation by picNuA4 and SAGA. Nucleic Acids Res. 2009;37(12):3969–3980.

    78. Collotta M, Bertazzi PA, Bollati V. Epigenetics and pesticides. Toxicology. 2013;307:35–41.

    79. Sutherland JE, Costa M. Epigenetics and the environment. Ann N Y Acad Sci. 2003;983:151–160.

    80. Gallou-Kabani C, Vige A, Gross MS, Junien C. Nutri-epigenomics: lifelong remodelling of our epigenomes by nutritional and metabolic factors and beyond. Clin Chem Lab Med. 2007;45(3):321–327.

    81. Miceli M, Bontempo P, Nebbioso A, Altucci L. Natural compounds in epigenetics: a current view. Food Chem Toxicol. 2014;73C:71–83.

    82. Duhaime-Ross A. Revved-up epigenetic sequencing may foster new diagnostics. Nat Med. 2014;20(1):2.

    83. van Berkum NL, Lieberman-Aiden E, Williams L, et al. Hi-C: a method to study the three-dimensional architecture of genomes. J Vis Exp 2010;(39):e1869.

    84. Khrameeva EE, Mironov AA, Fedonin GG, Khaitovich P, Gelfand MS. Spatial proximity and similarity of the epigenetic state of genome domains. PloS One. 2012;7(4):e33947.

    85. Hagarman JA, Motley MP, Kristjansdottir K, Soloway PD. Coordinate regulation of DNA methylation and H3K27me3 in mouse embryonic stem cells. PLoS One. 2013;8(1):e53880.

    86. Murphy PJ, Cipriany BR, Wallin CB, et al. Single-molecule analysis of combinatorial epigenomic states in normal and tumor cells. Proc Natl Acad Sci USA. 2013;110(19):7772–7777.

    87. Hyun BR, McElwee JL, Soloway PD. Single molecule and single cell epigenomics. Methods. 2015;72:41–50.

    Chapter 2

    Technologies for the Measurement and Mapping of Genomic 5-Methylcytosine and 5-Hydroxymethylcytosine

    Jolyon Terragni and Sriharsa Pradhan,    Genome Biology Division, New England Biolabs, Ipswich, Massachusetts, USA

    Over half of a century ago methylated cytosine in the genome was discovered. Since then progress has been made on the development of tools to study methylated cytosine. The first of these technologies utilized paper chromatography to measure genomic DNA methylation. Soon afterward researchers discovered that restriction enzymes and antibodies could also be used to detect and measure genomic methylation. Epigenetic technologies continued to evolve and enzymatic and chemical methods were proposed to study locus specific methylation. It was not until sometime later, during the genomics revolution, technologies were able to identify genome-wide methylation patterns. Recently, 5-hydroxymethylcytosine (5hmC) was rediscovered in the mammalian genome. Thereupon, it was found that some of these technologies could not distinguish between 5-methylcytosine (5mC) and 5hmC. This issue was resolved by modification of existing technologies, or creation of new approaches to study 5mC and 5hmC.

    Keywords

    DNA methylation; HPLC-mass spectrometry; bisulfite conversion; methylation-specific PCR; next-generation sequencing; Aba-Seq; Ox-BS; SMRT

    Chapter Outline

    2.1 Introduction 19

    2.2 Measuring Genomic Levels of 5-Methylcytosine and 5-Hydroxymethylcytosine 20

    2.3 Locus-Specific Analysis of 5-Methylcytosine and 5-Hydroxymethylcyotsine 23

    2.4 Technologies for Genome-Wide Analysis of 5-Methylcytosine and 5-Hydroxymethylcytosine 26

    2.4.1 DNA Microarray Approaches 26

    2.4.2 Next-Generation Sequencing Approaches 28

    2.5 Conclusions 34

    Acknowledgments 35

    References 35

    2.1 Introduction

    One of the most common and studied DNA modifications in the mammalian genome arises from the enzymatic methylation of cytosine to 5-methylcytosine (5mC) [1]. This cytosine modification, which is created de novo and is maintained by the DNA methyltransferase (DNMTs) family of enzymes, is critical in the regulation of gene expression [2,3]. Further illustrating the importance of this DNA modification, irregular patterning of cytosine methylation has been linked to many different diseases, such as acute myeloid and mixed lineage leukemias, AML and MLL respectively [4,5]. Until recently 5mC was thought to be the only mammalian DNA modification that exists, but new evidence acquired from human brain cells has revealed the presence of an additional DNA modification, 5-hydroxymethylcytosine (5hmC) [6]. 5hmC was initially identified as an oxidative DNA damage residue [7,8]. However, recent reports have demonstrated that the 5hmC modification arises by enzymatic oxidation of 5mC by the Ten-Eleven translocation (TET) family of enzymes [9]. Since the existence of 5hmC in the mammalian genome was a fairly recent discovery, researchers are still in the process of functionally characterizing this DNA modification. However, the current consensus among the epigenetic research community is that 5hmC is an essential intermediate in the active DNA demethylation pathway [9–13]. The epigenetic tools that have been created for the study of 5mC and 5hmC have focused solely on the measurement and mapping of these two epigenetic marks in the vertebrate genomes. In this chapter, we will discuss the creation and implementation of these different epigenetic technologies for the study of 5mC or 5hmC. Furthermore, each of these technologies will be discussed within the context of both cytosine modifications, since it is imperative to understand their ability to discriminate between 5mC and 5hmC.

    2.2 Measuring Genomic Levels of 5-Methylcytosine and 5-Hydroxymethylcytosine

    Methylated cytosine in mammalian genomes was originally discovered over half a century ago, in a study that utilized a paper chromatographic technique. In this foundational study, researchers first hydrolyzed genomic DNA isolated from the thymus of a calf and then separated the single nucleosides by paper chromatographic techniques, which revealed a fifth chromatography spot, identified as methylated cytosine [14]. As is often the case with a newly developed technology, there were several shortcomings with this method, such as the guanine not being completely hydrolyzed or the phenylalanine appearing on the paper in the region occupied by cytosine. It was not until 20 years later, when this assay was performed with ion pair–reversed phase high-pressure liquid chromatography (RP-HPLC), that it finally became the accepted standard for the measurement of genomic 5mC levels [15,16]. In the RP-HPLC method, an enzyme cocktail of DNase I, nuclease P1, and alkaline phosphatase is first utilized to digest genomic DNA down to single nucleosides. The individual pools of DNA bases are then separated through RP-HPLC and the individual bases measured for their UV absorbances (254 and 280 nm). Further improvements to this method have subsequently been made by incorporating mass spectrometry, downstream of standard HPLC nucleotide separation, to measure nucleotides with higher sensitivity and accuracy [17]. Time-of-flight and triple quadrupole mass spectrometers are two common instruments for the qualitative or quantitative analysis of modified cytosine nucleosides. The multiple reactions monitoring (MRM) method can detect 5mC and 5hmC at fmol level, thus making the discovery of low abundance cytosine modification possible [18].

    Over 50 years later after the initial discovery of 5mC, chromatographic and mass spectrometry techniques were also instrumental in the discovery of 5hmC in the mammalian genomes. In 2009, Kriaucionis et al. were initially trying to compare the abundance of genomic 5mC between Purkinje and granule cell nuclei, yet to their surprise they also discovered a sixth DNA base corresponding to hydroxymethylated cytosine [6]. In another study, published alongside this chromatographic study, it was also shown that the MLL Partner TET1 enzyme is responsible for the oxidation/conversion of 5mC into 5hmC [9]. Even though numerous improvements have been made through the years to the HPLC-mass spectrometry method for the measurement of modified nucleotides, there are some technical hurdles to this method that need to be considered when incorporating it into an epigenetic study. The most apparent limitation is the need for HPLC-mass spectrometry instrumentation and expertise, which can be problematic at research facilities with constrained resources. Additionally the HPLC-mass spectrometry method can take several days to complete, due to the requirement of genomic DNA having to be completely digested to single nucleosides before HPLC-mass spectrometry analysis. Lastly, this method is only capable of providing relative measurements of modified

    Enjoying the preview?
    Page 1 of 1