Epigenetic Gene Expression and Regulation

Epigenetic Gene Expression and Regulation reviews current knowledge on the heritable molecular mechanisms that regulate gene expression, contribute to disease susceptibility, and point to potential treatment in future therapies.

The book shows how these heritable mechanisms allow individual cells to establish stable and unique patterns of gene expression that can be passed through cell divisions without DNA mutations, thereby establishing how different heritable patterns of gene regulation control cell differentiation and organogenesis, resulting in a distinct human organism with a variety of differing cellular functions and tissues.

The work begins with basic biology, encompasses methods, cellular and tissue organization, topical issues in epigenetic evolution and environmental epigenesis, and lastly clinical disease discovery and treatment.

Each highly illustrated chapter is organized to briefly summarize current research, provide appropriate pedagogical guidance, pertinent methods, relevant model organisms, and clinical examples.

• Reviews current knowledge on the heritable molecular mechanisms that regulate gene expression, contribute to disease susceptibility, and point to potential treatment in future therapies
• Helps readers understand how epigenetic marks are targeted, and to what extent transgenerational epigenetic changes are instilled and possibly passed onto offspring
• Chapters are replete with clinical examples to empower the basic biology with translational significance
• Offers more than 100 illustrations to distill key concepts and decipher complex science

• Language: English
• Publisher: Academic Press
• Release date: Oct 19, 2015
• ISBN: 9780128004715

Book preview

Epigenetic Gene Expression and Regulation

Editors

Suming Huang

Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, Gainesville, FL, USA

Michael D. Litt

Center for Medical Education, Indiana University-Ball State University, Muncie, IN, USA

C. Ann Blakey

Department of Biology, Ball State University, Muncie, IN, USA

Table of Contents

Cover image

Title page

Translational Epigenetics Series

Copyright

Contributors

Preface

Acknowledgment

Chapter 1. Epigenetic gene expression—an introduction

1. Introduction

2. Epigenetics

3. Future directions and challenges

4. Summary

Chapter 2. Histone modifications—models and mechanisms

1. Introduction

2. Chromatin

3. The nucleosome

4. Histones and global mechanisms

5. Summary

Chapter 3. Genomic imprinting in mammals—memories of generations past

1. Introduction

2. The life cycle of imprinted regions

3. Mechanisms of genomic imprinting

4. Top-down dissection of imprinted domains

5. Transcriptional interpretation of the imprint

6. Imprinting and human disease

7. Finding new imprinted genes

8. The evolution of imprinting

9. Conclusion

Chapter 4. Polycomb and Trithorax factors in transcriptional and epigenetic regulation

1. Introduction

2. Regulators of homeotic genes—PcG and TrxG proteins

3. PcG-mediated gene repression

4. TrxG-mediated transcriptional regulation

5. Targeting of PcG/TrxG complexes to chromatin

6. Crosstalk with other PTMs

7. Regulation of PcG/TrxG function by upstream signaling pathways

8. Concluding remarks and future perspective

Chapter 5. Chromatin dynamics and genome organization in development and disease

1. Introduction

2. Technology advances in unraveling chromosome architecture and genome organization

3. Proteins involved in genome organization

4. Intra- and interchromosomal interactions during development

5. Intra- and interchromosomal interactions during carcinogenesis

6. Closing remarks and future directions

Chapter 6. ncRNA function in chromatin organization

1. Introduction

2. Small ncRNAs

3. Long ncRNAs

4. Noncoding RNA-based gene regulation by allostery

5. Summary

Chapter 7. Epigenetic gene regulation and stem cell function

1. Introduction

2. The 3D nuclear architecture of stem cells

3. DNA methylation in stem cells

4. Histone modifications in stem cells

5. Conclusive remarks on the importance of epigenetic-based methods to optimize the use of stem and progenitor cells for regenerative medicine

Chapter 8. Epigenetic inheritance

1. Introduction

2. Dynamics of epigenetic information throughout the life cycle

3. Epigenetic inheritance in ciliates

4. Epigenetic inheritance via DNA modifications

5. Epigenetic inheritance via histone modifications

6. Epigenetic inheritance through noncoding RNA

7. Prions

8. Unknown mechanism studies

9. Conclusions

Chapter 9. Transgenerational epigenetic regulation by environmental factors in human diseases

1. Introduction

2. Epigenetic regulations during early development

3. Environment and transgenerational epigenetic inheritance

4. Epigenetic diet and human diseases

5. Epigenetic inheritance of human diseases

6. Conclusions

Chapter 10. Identification of intergenic long noncoding RNA by deep sequencing

1. Introduction

2. An overview of methods for lncRNA detection

3. LincRNA identification based on RNA-seq

4. Regulation of lncRNA transcription

5. Future directions

Chapter 11. Regulation of erythroid cell differentiation by transcription factors, chromatin structure alterations, and noncoding RNA

1. Introduction

2. Erythroid cell differentiation

3. Hemoglobin

4. Heme synthesis

5. Red cell membrane

6. Regulation of gene expression during differentiation of erythroid cells

7. Conclusions

Chapter 12. Genetically altered cancer epigenome

1. Introduction

2. Histone modifications

3. DNA methylation

4. Nucleosome remodeling

5. Genome organization

6. Concluding remarks

Chapter 13. Long noncoding RNAs and carcinogenesis

1. Introduction

2. General features and mechanisms of action of the long noncoding RNA

3. Different functional strategies of lncRNAs

4. The long noncoding RNAs in cancer

5. Concluding remarks and perspectives

Chapter 14. Epigenetics of physiological and premature aging

1. Introduction

2. Epigenetics in aging: a broad outlook

3. Epigenetic mechanisms

4. Epigenetic regulation of cellular senescence

5. Convergence and divergence of epigenetic alterations to cause or control aging

6. Epigenetics and drug development

7. Conclusions

Chapter 15. Epigenetic effects of environment and diet

1. Introduction

2. Developmental origins of health and human disease

3. Epigenetic effects of environmental contaminants and toxins

4. Epigenetic effects of ethanol and tobacco exposure

5. Nutrition and DNA methylation

6. The Dutch famine

7. Maternal nutrition and fetal health

8. Folic acid and DNA methylation

9. Maternal nurturing and stress

Chapter 16. Epigenetic control of stress-induced apoptosis

1. Transcriptional regulation and apoptosis

2. Epigenetic control of proapoptotic genes during animal development

3. Epigenetic suppression of stress-responsive proapoptotic genes during tumorigenesis

4. Epigenetic regulation of anti-apoptotic genes

5. Epigenetics-based therapeutic strategies and derepression of proapoptotic genes

Chapter 17. Structure, regulation, and function of TET family proteins

1. Introduction

2. Structure of TET2

3. Enzymatic activity of TET family enzymes

4. Specificity of TET2

5. Posttranslational modification of TET2 and its interaction with other proteins

6. Regulation of TET2 activity

7. Regulation of TET2 expression

8. Biological function of TET2

9. TET2 gene mutations in hematological malignancies

10. TET2 gene mutations and solid tumors

11. Cooperation of TET2 gene mutation with other oncogenic mutations

12. TET2 and IDH1/2 gene mutations

13. Prognostic impact of TET2 gene mutations

14. Conclusion

Chapter 18. Epigenetic drugs for cancer therapy

1. Epigenetic pathways targeted for cancer therapy

2. Epigenetic drugs in clinic and clinical trials

3. Combined therapy

4. Closing remarks and future perspectives

List of acronyms and abbreviations

Glossary

Index

Translational Epigenetics Series

Trygve O. Tollefsbol, Series Editor

Transgenerational Epigenetics

Edited by Trygve O. Tollefsbol, 2014

Personalized Epigenetics

Edited by Trygve O. Tollefsbol, 2015

Epigenetic Technological Applications

Edited by Y. George Zheng, 2015

Epigenetic Cancer Therapy

Edited by Steven G. Gray, 2015

DNA Methylation and Complex Human Disease

By Michel Neidhart, 2015

Epigenomics in Health and Disease

Edited by Mario F. Fraga and Agustin F. F Fernández

Copyright

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Contributors

Blake Atwood,     Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA

Aissa Benyoucef

The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada

C. Ann Blakey,     Department of Biology, Ball State University, Muncie, IN, USA

Marjorie Brand

The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada

Jason O. Brant,     Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, University of Florida College of Medicine, Gainesville, FL, USA

Jörg Bungert,     Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida, USA

Yun Chen,     Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

Changwang Deng,     Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, USA

Nora Engel,     Fels Institute for Cancer Research, Temple University School of Medicine, Philadelphia, PA, USA

Alex Xiucheng Fan,     Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida, USA

Ekaterina Gavrilova,     Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida, USA

Shrestha Ghosh,     Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

Keith E. Giles,     Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA

Suming Huang,     Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, USA

Mir A. Hossain,     Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida, USA

Gangqing Hu,     Systems Biology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

Xin Hu,     Edmond H. Fischer Signal Transduction Laboratory, School of Life Sciences, Jilin University, Changchun, China

Wei Jian,     Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

Alta Johnson,     Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

Priscilla Nga Ieng Lau,     Leukemia and Stem Cell Biology Group, Department of Haematological Medicine, King’s College London, London, UK

Michael D. Litt,     Center for Medical Education, Indiana University-Ball State University, Muncie, IN, USA

Xuehui Li,     Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

Yangqiu Li,     Institute of Hematology, Jinan University Medical College, Guangzhou, China

Yuanyuan Li

Department of Medicine, Division of Hematology and Oncology, University of Alabama at Birmingham, Birmingham, AL, USA

Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA

Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, AL, USA

Xiumei Lin,     Department of Hematology,Guangzhou First People’s Hospital,Guangzhou,China

Jianrong Lu,     Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA

Huacheng Luo,     Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA

Christine M. McBride,     Department of Biology, The University of Alabama at Birmingham, Birmingham, AL, USA

Benjamin B. Mills,     Department of Biology, The University of Alabama at Birmingham, Birmingham, AL, USA

Bhavita Patel,     Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA

Yi Qiu,     Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

Félix Recillas-Targa,     Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México, Ciudad de México, México

Nicole C. Riddle,     Department of Biology, The University of Alabama at Birmingham, Birmingham, AL, USA

Chi Wai Eric So,     Leukemia and Stem Cell Biology Group, Department of Haematological Medicine, King’s College London, London, UK

Jared Stees,     Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, Florida, USA

Ming Tang,     Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, USA

Jessica L. Woolnough,     Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA

Bowen Yan,     Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

Thomas P. Yang,     Department of Biochemistry and Molecular Biology, Center for Epigenetics, Genetics Institute, University of Florida College of Medicine, Gainesville, FL, USA

Yurong Yang,     Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida, USA

Keji Zhao,     Systems Biology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA

Zhizhuang Joe Zhao

Edmond H. Fischer Signal Transduction Laboratory, School of Life Sciences, Jilin University, Changchun, China

Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

Lei Zhou,     Department of Molecular Genetics and Microbiology, UF Health Cancer Center, College of Medicine, University of Florida, Gainesville, Florida, USA

Zhongjun Zhou

Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China

Shenzhen Institute of Research and Innovation, The University of Hong Kong, Shenzhen, China

Preface

The definition of epigenetics has changed a number of times over the past decades, reflecting our increasingly detailed understanding of regulatory mechanisms in eukaryotes. The chapters of this book effectively point the way to a contemporary definition. Epigenetics, as defined in recent years, concerns itself with the transmission through cell division, and in some cases through the germline, of phenotypic information that is not contained in the DNA sequence itself. The search for biochemical mechanisms that could implement an epigenetic program has, reasonably, focused on reactions that lead to covalent modifications of DNA, and on the identity and biochemistry of proteins and nucleic acids that might bind to DNA tightly enough to remain attached through cell division. That search initially focused on DNA methylation, and more recently on the nucleosome, the octamer histone complex that binds tightly to DNA and packages virtually all of the genome.

DNA methylation provides potentially the most straightforward mechanism for epigenetic transmission of information. As first pointed out over 40  years ago, cytosine methylation at CpG sites can be propagated during replication by an enzyme that recognizes the hemimethylated site and methylates the CpG on the newly synthesized strand. Subsequent studies, notably at imprinted loci, have shown that DNA methylation can affect, directly or indirectly, the binding of transcription factors. The DNA methylation model, in fact, provided the impetus for the present definition of epigenetics, because it appeared to represent the first plausible pathway for transmission through DNA replication of information not encoded in DNA sequence. Since then, however, discovery of the role of chromatin structure in regulation of gene expression, and, during the last twenty years, of the role of noncoding RNAs, has greatly expanded the scope of the epigenetic enterprise.

A large number of articles in this book examine the now extensive literature on the relationship between chromatin structure and gene expression. At the level of individual nucleosomes, the obvious first question is whether nucleosome stability and placement play an important regulatory role. One major research direction has addressed the identity, biochemical consequences, and biological function of histone covalent modifications. The identified distinct modifications include acetylation, mono-, di-, or trimethylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, and more. Each modification targets specific residues on the individual histones of the nucleosome, but the effects, it is now clear, cannot be described in terms of a single code. As with other regulatory mechanisms in eukaryotes, organisms appear to take advantage of the combinatorial versatility afforded by the wide variety of modifications. As described in several chapters, these modifications are an integral part of mechanisms that control initiation of transcription, transcriptional pausing, transcript elongation, termination, and RNA splicing. Other modifications are involved in DNA replication and DNA damage repair. Some marks are predominantly associated with transcriptionally active or inactive chromatin. Certain combinations of marks tend to appear at promoters, others at enhancers. In pluripotent stem cells, some promoters carry both activating and silencing modifications, in principle making the cells ready for rapid changes in expression, depending on the developmental path they take. Histone modifications also play a critical role in transcriptional elongation. When RNA polymerase II transcribes a gene, it carries with it histone modifying enzymes, signaling the formation of a chromatin structure that discourages inappropriate initiation of transcription from within the gene coding region.

All of these reactions are carried out by an array of enzyme complexes specialized to add or remove the individual modifications at particular sites on the histones. Typically, these complexes carry, in addition to the subunit containing the active site, a variety of other protein cofactors that can confer further target specificity. Notable among these modifying complexes are the Polycomb and Trithorax groups, associated respectively with transcriptional silencing and activation. Other complexes are devoted to the task of removing modifications: enzymes and pathways have been identified not only for deacetylation, but for removal of methyl groups from histone lysine and arginine residues. Modifications are thus dynamically regulated in response to regulatory signals and are an essential part of the mechanism by which genetic information contained in DNA is selectively expressed.

How are these modifications used to transmit that information? Modified histones can, in some cases, recruit transcription factor complexes to promoters and enhancers. Other large families of protein complexes use ATP to move nucleosomes away from important DNA regulatory sites or to position them so that they cover these sites. Some histone modifications can alter the strength of interaction between histones and DNA. Certain modified histones can recruit enzymes involved in DNA methylation, and 5-methylcytosine can recruit histone modifying enzymes, thus coupling the two kinds of potentially epigenetic signals. In every case, the protein complexes involved can vary in details of subunit content and specificity, making it necessary to study each regulatory pathway as a separate problem. This has become especially clear as we have begun to understand the role of noncoding RNAs (ncRNAs) in regulatory processes and especially in epigenetic mechanisms involving chromatin. In particular, ncRNAs help to deliver Polycomb or Trithorax group complexes to specific sites, thus guiding the delivery of silencing or activating marks. Other ncRNAs function as part of regulatory protein complexes, and are essential for their activity.

Although much of this work has focused on epigenetic effects at the level of individual genes or gene families, recent studies have revealed the importance of large scale organization within the nucleus in the control of gene expression. It has long been known that enhancers can in some instances act over very great distances to activate specific promoters. It has not been clear how the enhancer chooses its target. Chromatin conformation capture technology (3C, 4C, 5C, and Hi-C) has revealed the presence of large scale organization of the genome into loop domains, with interactions within loops favored over those between loops. How such loops are established and maintained through cell division is a question that remains to be explored and may well be an important part of the epigenetic machinery.

One question raised by all these mechanisms is the extent to which they fit the present definition of epigenetics. There is, at least in principle, a way for DNA methylation patterns to be transmitted through cell division, as described above. Patterns of histone modifications could also be preserved: for example, the Polycomb complex, PRC2, which methylates lysine 27 on histone H3, can be recruited specifically to histones carrying that mark, and could then modify adjacent histones newly deposited at the replication fork. Similarly, one could propose that some part of the loop domain organization of the genome is maintained, although it is known that many features of this higher order structure are not preserved during mitosis. Demonstrating the actual use of such mechanisms in vivo has, however, been difficult, particularly for those mechanisms involving chromatin structure. To qualify according to the strict definition, chromatin structure at a gene should be preserved through cell division and should be determinative of the state of activity of that gene in the daughter cells. From one point of view, changes in histone modifications or nucleosome position could be viewed simply as a part of the mechanism of gene expression–a consequence, rather than a cause of the activity state. For example, an active gene could be maintained in that state because relevant transcription factors remain at high concentrations through mitosis. Afterward, these factors might be sufficient to reestablish activity, and appropriate histone modifications could be regenerated.

Because it is difficult to distinguish between cause and effect, there has been resistance to the idea that chromatin structure and histone modifications convey epigenetic information. Given their evident significance for cell function, this is perhaps not such an important objection. As has been suggested before, it may be more appropriate to revise the definition of epigenetics, which in earlier times referred to the developmental processes that led from a single fertilized egg to a complete organism. There can be no doubt about the role of histones, chromatin, and DNA methylation in those processes. At the same time, it should be clear that mechanisms do exist for inheritance of information that is not carried in the DNA sequence. Position effect variegation in Drosophila provided the first evidence for a change in phenotype that was connected with location of a gene within a chromosome, rather than with changes in the gene itself. A clear example exists even in Saccharomyces, where genes located near telomeres can be maintained in a silent state through many generations, then switch to an active state, which is likewise stable through many cell divisions. In neither case is there any change in the DNA sequence itself; changes in chromatin structure provide the necessary signals.

Although other examples of transmission through cell division exist, epigenetic inheritance through the germline has been more difficult to demonstrate. A clear example is provided by the agouti mouse, in which coat color, determined by DNA methylation patterns of a retrotransposon inserted near the agouti gene, is transmitted to offspring. Other examples exist in plants. There is also evidence for multigenerational epigenetic transmission of phenotypes in flies, mice, and humans. It should be noted that the fidelity of transmission is not as high as for genetic inheritance. However to the extent that these phenotypic changes help to stabilize certain mutations, they may contribute to more permanent changes marked directly in the genome. Because DNA methylation and probably chromatin chemistry can be influenced by environment (e.g., diet), epigenetically controlled phenotypes can reflect environmental signals. The old arguments about heredity vs. environment can now be seen in a new light. But whatever the contribution of these epigenetic signals to inherited phenotype, their importance transcends definitions. They are the manifestations of the array of biochemical pathways that modulate all DNA function in eukaryotes and make possible the enormous variety of cell behaviors required for the success of multicellular organisms.

Gary Felsenfeld,     Laboratory of Molecular Biology National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD, USA

Acknowledgment

This work was supported by the intramural research program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases.

Chapter 1

Epigenetic gene expression—an introduction

C. Ann Blakey¹,  and Michael D. Litt²     ¹Department of Biology, Ball State University, Muncie, IN, USA     ²Center for Medical Education, Indiana University-Ball State University, Muncie, IN, USA

Abstract

Epigenetics research encompasses a wide range of areas, from basic research on chromatin structure to examination of growth and development of the organism, to investigations of the development and progression of complex diseases. Along the path, epigenetic research has developed tools and technologies that facilitate the rapid progression of discoveries and is now approaching a point of implementation. Presented is an overview of the ideas and range of current biomedical epigenetic research efforts underway, as well as a brief discussion of a few key issues hindering progress.

Keywords

Biocomputational tools; Chromatin; Epigenetics; Histone modifications; ncRNA; Nomenclature; Software algorithms

Contents

1. Introduction 1

1.1 Epigenetics – a unifying concept for genetic mechanisms 1

1.2 Ideas and questions 2

2. Epigenetics 3

2.1 Regulating the process 3

2.2 DNA methylation 3

2.3 Chromatin 4

2.3.1 Histone modifications 4

2.3.2 Chromatin organization and remodeling 5

2.4 RNA 6

2.5 Clinical implications 7

3. Future directions and challenges 8

3.1 Analytical tools 8

3.2 Databases 11

3.3 Nomenclature 11

4. Summary 13

4.1 Core concepts of the text 13

4.2 Final thoughts 15

Acknowledgments 16

References 16

1. Introduction

1.1. Epigenetics – a unifying concept for genetic mechanisms

One could argue that every aspect of a person’s health is influenced by the expression of their genes. There are approximately 20,000 protein-coding genes, though a seemingly ever-changing number, controlling the fate and development of cells within the human organism. This cacophony of differential gene expression influences an organism from the point of initial development upon fertilization, onward through each stage of growth and maturation, and includes all interactions—both genetic and environmental—at each of these stages. Consequently, understanding the mechanisms that control and regulate gene expression is pivotal to expanding our knowledge and tools to manipulate the health of the human organism. As a result, interpretation of genetic and environmental data, and how best to analyze and utilize that data, has become a key focus area in biomedical research. The implications for the development and application of treatments in clinical medicine are still in their infancy. However, current studies targeting diseases, nutritional-based issues, and environmental-associated damage have made remarkable advances through the use of the tools of genomics and bioinformatics, revealing a convergence into the field known as epigenetics.

At the birth of genetics, the underlying mechanisms of these processes may have eluded us; nonetheless, observational data analysis has led to significant discoveries tying the various fields of biological science together. In his 2014 review, A Brief History of Epigenetics, Felsenfeld [1] provides an excellent synthesis and perspective of epigenetics, from both a historical and theoretical view of genetics. Epigenetics was originally used to describe the processes involved in the development of a zygote to a mature organism. Through a clear retrospective description of our stepwise progression from brilliant insights and clouded uncertainties to our current level of understanding, Felsenfeld [1] weaves together the rich tapestry that is our current understanding of epigenetics. The depth of epigenetics’ role becomes visible through the examination of the landmark discoveries and significant contributions of researchers across a range of organisms and questions. Past research discoveries allowed for an examination of connections and interactions from the gene to cellular to organismal levels. Molecular and genomic studies now allow for further refinement and redefining on all levels. A new perspective is therefore needed in order to consider the active role of genes, genomes, and gene/genome expression and interactions in the health of the organism and its future offspring. Thus, we now find ourselves looking upon a new epigenetic horizon with a far more complex landscape, encompassing and reaching beyond that originally envisioned by Waddington [2–6].

1.2. Ideas and questions

Many ideas explored in this text can be approached as questions, though more arise to the reader; most are yet to be fully resolved:

1. How does chromatin change and evolve, not just in the sense of evolution?

2. What are the combinatorial effects of these modifications on the chromatin?

3. How is methylation (DNA, histones, or other) coordinated to provide control across cell generations?

4. In what role(s) are RNAs critical to the models of epigenetic regulation?

5. What are critical protein complexes critical to epigenetic processes?

6. How are these critical epigenetic protein complexes structured and regulated?

7. How does an epigenetic system develop and is regulated in an organism?

8. How does an epigenetic system change/evolve within the organism, including modification and regulation within the vast array of tissues and activities?

9. How is epigenetics research changing biomedical approaches to disease?

10. How is the concept of transgenerational epigenetics, including environment and nutrition effects, changing our view of biology and genetics?

Initially, when viewing the human organism, we see the physical features that comprise the unique characters that identify one individual from another and differentiate the human organism from other species. As we delve deeper into the molecular nature of a single organism, we find that the genetic makeup (hereditary material) of the organism and each cell, with the exception of gametic cells, is relatively identical. However, it is the relative identical nature that keeps the organism from being one homogenous mass of cells. Thus, we arrive at a very old question: how does the organism take a single set of instructions and derive the vast array of different tissue types and organs and organ systems that comprise a typical functioning organism [7]?

Thus, when we examine a eukaryotic organism beginning with the gamete, all of the key processes in the organism’s life cycle must be tightly regulated both temporally and spatially. These regulatory mechanisms must monitor and control all the critical processes within the organism of the current generation (beginning with sperm and egg) through to its ultimate senescence and death and include the production of the next generation, as well. These processes include all associated biological activities involved in fertilization, zygote formation, and growth and development of the embryo, and growth, development, and maturation of the adult organism. Therefore, the magnitude of the role of epigenetics, and subsequently transgenerational epigenetics, is just beginning to be understood [8].

2. Epigenetics

2.1. Regulating the process

To understand the role epigenetic mechanisms play in the life cycle of an organism, we must consider the wide range of different types and categories of molecular structures at work within a cell and organism, where the major categories are DNA, RNAs, and special classes of proteins. We must consider the mechanisms capable of modifying the initial set of information delivered by the sperm and egg in the formation of the zygote, to the functional sets found in each of the tissues, organs, and systems that comprise the organism at each stage of development. Epigenetics mechanisms provide not only a way but also a means by which these modifications can occur without the need for alteration of the original DNA sequence of the organism [1]. The resulting temporal and spatial expression of the genetic information allows for the appropriate growth and development of a healthy organism.

2.2. DNA methylation

Altering the organism without changing the primary sequence of the DNA can be accomplished through several mechanisms. One category of these mechanisms involves the chemical modification of deoxycytidine residues within specific cytosine-guanine-rich sequences, typically via methylation and/or demethylation, but others include hydromethylation and the derivatives of demethylation, formylation, and carboxylation [9]. Renewed and growing interest in the potential role of highly conserved modification to deoxyadenine residues, once relegated by many as mechanisms solely of prokaryote systems, has attained a stature as an important mechanism in spatiotemporal regulation of eukaryotic development [9–11]. These modifications to the DNA sequence have the potential to both alter the three-dimensional structure of the molecule of the DNA strand and inhibit or allow for accessibility to the DNA structure by other molecules. Accessibility, in turn, plays a role in gene expression and therefore, the ability of the cell to perform necessary functions at critical junctures in the organism’s life cycle [12].

In an oversimplified example, the methylation of the fifth carbon in the cytosine base of a cytosine-guanine-rich promoter region can result in limited accessibility by the transcription machinery [12]. In the converse case, the demethylation of these 5-methylcytidine residues results in the previously inaccessible region becoming accessible to regulatory factors. This may subsequently promote active expression of the downstream gene versus repressing it. Thus, methylation has effectively silenced the gene. While this is a highly oversimplified example, it serves to emphasize the point that methylation and demethylation do not alter the sequence of the DNA yet still have the potential to dynamically change the accessibility of the structure and expression of gene sequences [13–15]. Elegant examples, with far greater descriptions of mechanisms associated with imprinting and embryonic development and disease progression, are presented in this volume chapters 3, 7, 8, 15, 17, and 18.

2.3. Chromatin

2.3.1. Histone modifications

When considering epigenetics modifications, methylation and demethylation of DNA are the first mechanisms that must be understood. Recognition of DNA methylation and demethylation, alone, does not provide a sufficiently complete explanation of the epigenetic phenomena at work; therefore, researchers must consider the regulation of the genome from the larger context of chromatin structure. Here, chromatin structure takes on additional levels of complexity that can begin to account for some of the observed regulator mechanisms at work in the genome. This complexity includes both double-helical DNA linker regions (upto ~90 bp in length), and regions composed of both double-helical DNA and histone proteins called a nucleosome. More precisely, the nucleosome is composed of approximately 147 bp of double-helix DNA coiled 1.65 times around a histone octamer protein core [16,17].

The histone octamer component of the nucleosome, as an independent structure relative to the double-stranded DNA molecule, is of critical importance in the epigenetic control of the genome [18–21]. This unique, eight-subunit structure is composed of two units each of the histone proteins H2A, H2B, H3, and H4 [16,18]. A fifth histone protein, H1, is not part of the nucleosome core particle, but retains important functional roles within the structure and dynamics of chromatin [22–24]. Variant forms of the octamer core exist and are intimately associated with specific regions of the genome [25,26].

The main four posttranslational modifications of the nucleosome proteins, methylation, acetylation, phosphorylation, and ubiquitination, are most often discussed in textbooks. But there remain a host of other posttranslational modifications (Table 1) that can occur, which, depending upon the temporal and spatial location of the nucleosome, will have different effects in different cell types during the developmental program. The octamer can have far-reaching effects on the overall organization and structure of the DNA to which it is associated, including the regulation of access to the double-stranded DNA molecule such as by methylation/demethylation of the DNA or by transcriptional machinery of the nucleus. The specific arrangement and chemical modifications of each of these proteins can have a major impact on gene expression and thus, the well-being of the organism [25–36]. These additional forms of histone modifications, as well as new forms, require further exploration and research.

2.3.2. Chromatin organization and remodeling

The nucleosome and linker regions of DNA coil into different hierarchical levels of organization, depending upon the histone variants of the region and the modifications to the DNA and the histone components of the nucleosomes. Together, these components of chromatin function as integral components of the genome or more precisely, when contemplated relative to their coordinated roles in gene expression, the epigenome.

Table 1

Major histone modifications, not all-inclusive

Acetylation

ADP-ribosylation

Butyrylation

Citrullination

Crotonylation

Formylation

0-GlnNAc-glucosamine (0-GlcNAc)

Glutathionylation

Glycosylation

Hydroxylation (or 5-Hydroxylation)

Malonylation

Methylation

Phosphorylation

Proline isomerization

Proprionylation

Succinylation

Sumoylation (similar to ubiquitination)

Ubiquitination

Excellent publications on chromatin structure, organization, and remodeling have begun to appear with great regularity. Current literature of the biosciences has become dominated by investigations into possible connection of epigenetics to disease and cancers. These works have significantly impacted our ability to commence to understand the depth and range of mechanisms of histone and chromatin modifications that is possible [37–41] and therefore, their subsequent contributions to chromatin dynamics and remodeling within the genome [33,42–46].

Extensive discussions on histone modifications, chromatin dynamics and remodeling can be found in this volume in chapters 3, 4, 5, and 14.

2.4. RNA

As with any biological process, the correct timing of gene expression will play a pivotal role. Facilitating the expression of genes and the mechanisms that allow for expression are a host of molecules, involving a wide range of ribonucleic acids (RNA, see Table 2). The roles of these RNAs make it possible for the myriad of cascading processes necessary for homeostasis, as well as the adaptive nature of the organism to survive environmental stresses. RNAs are involved in the methylation of DNA [47], as well as having other structural, catalytic, and regulatory functions within chromatin modification machinery [48,49]. Detailed discussions of the various mechanisms associated with epigenetics and different RNAs can be found in this volume in chapters 3, 6, 10, 11, and 13.

Table 2

Some classes of RNAs involved in epigenetics gene expression, not all-inclusive

2.5. Clinical implications

Through the study of epigenetics, we now begin to see the connections to biological processes that had heretofore remained a puzzle to medical science. In many ways, we are at the infancy of where epigenetic research may take biomedical research as we begin to elucidate the full range and coordination of the epigenetic mechanisms at work in eukaryotic organisms. Our understanding of these processes continues to expand as we examine the disturbances observed in utero cells undergoing differentiation, to the establishment of imprinting patterns within the sperm genome contribution of a newly fertilized egg, or the modifications of histones during X-chromosome inactivation. These topics are discussed in greater detail in this volume in chapters 3, 7, 8, 11, and 13.

Historical records can provide important data in epigenetic discovery, as has been clearly shown by the example of Dutch Hunger Winter analyses of health data of subsequent generations within that population [50]. This famine occurred between September/October 1944 and May 1945 and ended with the end of World War II. A total of 18–22,000 deaths were associated with this famine, although a total of 4.5  million individuals were actually affected by a 400–800 calorie per day diet [51]. "Studies that draw upon multigenerational data sets allow for the analyses of extremes of environmental influences that cause resultant disturbances in the primordial germ cells of an organism. In the case of the Dutch Hunger Winter, researchers were able to follow epigenetic changes in several generations. For example, the women (F0 generation) who carried female progeny (F1 generation) in gestation during the months of the Dutch Hunger Winter, as well as follow those changes in the subsequent generation of in utero primordial germ cells (F2 generation) [52]. The transgenerational epigenetics effects of the Dutch Hunger Winter are discussed in greater detail in this volume in chapter 15.

Evaluation of changes in patterning that differ from those who did not undergo an environmental or nutritional stress, and the persistence of pattern alterations, not just DNA methylation but also modified nucleosomal patterning, add greater depth to our knowledge and understanding of the impact of these long-term stresses. The ramifications of alterations in the genetic mechanisms at work have become an area of intense research in the biomedical sciences [53,54]. Companies collaborating with public–private partnerships have established one of the largest joint ventures of its kind, called the EpiGen Consortium (www.epigengrc.com/).

When gene expression does not follow the expected program or when a disease develops, understanding the epigenetic changes associated with these disease states may help further our ability to develop clinical approaches to the treatment of diseases. Explorations of epigenetic phenomena associated with various cancers and diseases (for examples, see Table 3) add emphasis to the relationship between the fine level of regulation of chromatin structure, accessibility, regulation of gene expression, and the transcriptional machinery. Detailed discussions and analyses of the role of epigenetics in cancers and disease can be found in this volume in chapters 9, 12, 14, 16, and 18.

Table 3

Some epigenetic-influenced diseases, syndromes, and cancers, not all-inclusive

3. Future directions and challenges

3.1. Analytical tools

The quantity and quality of data produced in epigenetics has increased exponentially since the 2010. The vast array of techniques and technologies that were initially developed to further enhance our ability to rapidly sequence the genome and its products has led to a need for greater bioinformatics and biocomputational capabilities to handle the large, nearly overwhelming volumes of resultant data (Tables 4–6). The difficulty arises when attempting to source the best tools for a specific application from the range of tools available, as well as the location or deposition of these tools for utilization in the case of software algorithms and packages.

As excellent as these resources have proven to the discovery of new data relationships, there remains an issue of complete interconnectedness across databases. Valuable time and labor is lost without continual improvements in workflows for automated data handling, processing, and database access. These latter challenges focus primarily on software and hardware communications. Thus, there will remain a continual need for more highly trained researchers and personnel with up-to-date knowledge and skills in both computer science and the biomedical sciences.

Table 4

Molecular techniques utilized in epigenetic studies, not all-inclusive

Table 5

Biocomputational algorithms for genomic analyses, not all-inclusive

Table 6

Biocomputational algorithms for transcriptomics analyses, not all-inclusive

Identification of appropriate computational/biocomputational tools, even if freely available for use to researchers, often requires a continual surveillance of the bioinformatics literature in order to remain up-to-date on the newest, or to find the latest developments. These tasks have become even more relevant, both in their use within the biomedical/biological field of specialization and as biocomputational tools.

Many researchers have attempted to facilitate and share their own interconnectivity with data and data workflows with their colleagues through the development of Web sites and database structures which, in turn, cross-reference the multiple external databases, algorithms, and resources utilized in their own endeavors. But, these approaches become cumbersome and maintenance of computer links are labor intensive and withdraw resources from the primary funded areas of their research projects. Resources for research are a scarce commodity, and highly trained personnel is an ever-increasing expense. So, the impetus to create more efficient workflow algorithms and data handling methodologies will continue. As with most areas of genomics, technology for data production far exceeds our ability to process it, and backlogs are created. The improvement of workflow will remain a constant battle as the deluge of data continues to mount. It is admirable that so many researchers share their tools and approaches beyond publications. However, the challenges of accessibility and integration for improved workflow without loss of researcher productivity still remain key issues that have yet to be resolved.

3.2. Databases

The major databases and portals such as NCBI, EMBL, GenBANK, ENCODE, and GENCODE have provided a significant venue for researchers to access large volumes of data and associated information beyond that which individual research projects can provide. Recognition of the need for continual work toward integration of cytogenetic, molecular, and cellular data for more rapid discovery and application of knowledge in the biomedical sciences remains clearly evident.

There are numerous individual databases available to the researchers interested in pursuing epigenetic studies or simply wishing to understand the interconnections of available genomic information (Table 7). Through portals, it is possible to find a vast array of information and bioinformatics tools. Online datamining resources have become much more accessible, such as YeastMine (yeastmine.yeastgenome.org), MouseMine (www.mousemine.org), FlyMine (www.flymine.org), or metabolicMine (www.metabolicmine.org), to name only a few, all of which allow for rapid access to depths of information both within and across species.

No one portal or database can provide all the resources currently required by the scientific community. In addition, changes in funding can dramatically alter or even curtail accessibility to key database resources for the entire research community. The fields of bioinformatics and biocomputational analyses continue to work diligently toward greater accuracy and efficiencies, but continual funding for maintenance, improvements, and new developments in database structure, automation, and accessibility will remain key issues.

3.3. Nomenclature

One of the difficulties in contemplating the enormous volume of research that is being produced in this rapidly expanding area is that of nomenclature. Cross-species nomenclature issues slow discovery when every alias of a particular gene or transcript needs to be searched using multiple search engines or mining algorithms in order to obtain the fullest degree of information on known or putative functions and interactions. While some researchers do provide tables with regard to their particular transcripts or genes of interest when they publish in order to facilitate the reader and future discovery, these are not typical. Some authors in various publications have provided tables of a limited number of homologs within their publication to improve cross-species access to important findings.

Table 7

Some key databases, from which additional data-mining tools and resources can be accessed

Still, there remains a significant issue with regards to the acronyms and cross-referencing of genes and gene fragments. An example of the problem can be illustrated with the Trithorax group protein (trx) from Drosophila, where the human homolog is KMT2A with the aliases HTRX1, HRX, MLL, and MLL1 (Table 8). As research has progressed, there have been greater efforts to keep the same acronym across species. We find many of the homologous genes in humans and mouse, with the exception that the human acronym is all capital letters, and the mouse has only the first letter capitalized. In the case of the human gene aliases TRX, TRX1, and TRDX, these acronyms are for the cytoplasmic thioredoxin gene (TXN), which is also known as TRX1 and LMA1 in Saccharomyces and TRX2 in Drosophila.

Therefore, clear identification of the organism(s) and the primary gene name will help reduce some of the sources of confusion in literature and discussions. Thus, greater clarity of the true breadth of data coverage in this field will become more apparent and accessible, and facilitating faster discovery and utilization of information through striving with care to avoid the use of secondary aliases and non-organism-specific acronyms, identifying the organism in question within discussions, and continuing to provide cross-references to key model organisms where possible.

4. Summary

4.1. Core concepts of the text

The chapters in this text are arranged to provide the reader both an overview of current developments in this exciting and dynamic field of research and to function as a resource for graduate students and biomedical professionals.

In the first part of the text, the reader will be presented with an overview, as well as in-depth reviews of major components of epigenetics research. A review of the current histone modifications models is presented. Next, a discussion on chromatin dynamics and interactions provides additional depth. This is followed by a close examination of chromatin organization and the role of noncoding RNA in epigenetics. Subsequent chapters focus on the tools of epigenetic research and their application, thereby providing readers a solid background from which to venture further.

In the second part of the text, the reader will be introduced to epigenetic effects from the basis of our understanding of its heritability to its role in imprinted-associated gene expression. Next, chapters present discussions of epigenetic mechanisms in stem cells and tissue development, which ultimately lead the reader to examine the effects of epigenetics on the aging process. Afterward, discussions consider the possible epigenetic effects on future generations (transgenerational epigenetics), and those of the environment.

Finally, in the third part of the text, discussions consider the role of epigenetics on a specific area of biomedical research—cancer epigenetics. Beginning with apoptosis, the reader is presented mechanisms of regulation and roles of epigenetics in cancer. The specific role of major protein complexes, such as the Polycomb group and Trithorax group proteins, is examined. These protein complexes, along with an array of noncoding RNAs, are considered throughout with regard to gene expression pathways associated with carcinogenesis and they provide expert insights into the development of new therapies.

Table 8

A comparison of the gene names and aliases is given for the Trithorax Group Protein (TRX-G) for Lysine (K) Methyltransferase 2A/B across 4 species versus the cytoplasmic form of the Thioredoxin (TXN) gene. In each case, the primary gene name is provided as well as several aliases, though every gene alias is not included. As shown in red, the gene name TRX, TRX1, and TRX2 are utilized across all species and within a species (Drosophila) for two distinctly different genes

a Trx2 (location: chrVII) in yeast has a paralog, TRX1.

b LMA1 is used both as aliases in yeast for TRX1, TRX2, and PBI2 and for the complex formed between TRX1 and Pbi2p.

c PBI2 (location: chrXIV)  =  Pbi2p, also known as I2B or IB2.

4.2. Final thoughts

E.B. Wilson made the following observation in 1895 [55]:

"Now, chromatin