Environmental Epigenetics in Toxicology and Public Health

Environmental Epigenetics in Toxicology and Public Health provides in-depth discussions of the suite of complex environmental factors shown to impact epigenetic components within the cell, as well as evidence that these epigenetic modifications are tied to early and later life health effects. This book offers a translational research perspective, highlighting both in vivo and human population-based evidence for ties between the environment, the epigenome, and health outcomes, with an emphasis on evidence for transgenerational effects of exposures, as well as developmental windows of susceptibility to environmentally-linked epigenetic effects.

This volume in the Translational Epigenetics series aides in the development of new therapeutic options meant to reverse inappropriate epigenetic alterations, helping researchers in their efforts prevent and treat a variety of chronic diseases tied to environmental exposures.

• Offers a thorough discussion of the environmental factors influencing epigenetic mechanisms in early and late life, and in transgenerational inheritance
• Examines both animal model and human population-based research in environmental epigenetics, highlighting developmental windows of vulnerability to epigenetic modification
• Features contributions from international experts in the field

• Language: English
• Publisher: Academic Press
• Release date: Oct 18, 2020
• ISBN: 9780128199695

Book preview

Environmental Epigenetics in Toxicology and Public Health

First Edition

Rebecca C. Fry

Carol Remmer Angle Distinguished Professor, Department of Environmental Sciences and Engineering, Institute for Environmental Health Solutions, Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Series Editor

Trygve Tollefsbol

Professor of Biology, Senior Scientist, Comprehensive Cancer Center, Comprehensive Center for Healthy Aging, University of Alabama at Birmingham, Birmingham, AL, United States

Table of Contents

Cover image

Title page

Copyright

Contributors

Preface

Acknowledgments and Dedication

Section I: Epigenetic mechanisms/machinery

Chapter 1: Epigenetics: An overview of CpG methylation, chromatin remodeling, and regulatory/noncoding RNAs

Abstract

Introduction to epigenetics and its application to the central dogma of biology

CpG methylation

Chromatin remodeling

Regulatory/noncoding RNAs

Concluding summary

Chapter 2: Tools for the assessment of epigenetic regulation

Abstract

Introduction

Brief history of the development of technologies to assess the epigenome

High-throughput sequencing

Microarrays

Three key differences in approaches in the design and analysis of transcriptomics experiments

Tools to assess CpG methylation

Tools to assess chromatin structure and regulation

Tools to assess miRNA and other small noncoding RNAs

Pathway analysis

Conclusion

Section II: Transdisciplinary approaches for the study of environmental epigenetics

Chapter 3: Model organisms and their application in environmental epigenetics

Abstract

Introduction

Vertebrates

Invertebrates

Plants

Unicellular eukaryotes

Conclusions

Chapter 4: Epidemiological concepts in environmental epigenetics

Abstract

A conceptual framework for epigenetics in environmental health and epidemiology

Considerations for the design and conduct of epigenetic epidemiology studies

Inferences about causality

Conclusions and future directions

Section III: Epigenetic programming of disease in relation to the environment

Chapter 5: Pregnancy and birth outcomes: A role for environment-epigenome interactions

Abstract

Early pregnancy insults

Later pregnancy insults

Summary

Chapter 6: Neurodevelopment outcomes

Abstract

Introduction

Organic compounds

Inorganic compounds and neurodevelopmental outcomes

Summary

Chapter 7: Cancer

Abstract

Disclaimer

Introduction

Part 1: Pharmacological exposures

Part 2: Ionizing radiation

Part 3: Lifestyle and personal behavior

Part 4: Infectious agents

Part 5: Proxies for early-life environmental exposures: Birthweight

Conclusion

Chapter 8: Epigenetics and environmental programing of lung disease

Abstract

Introduction

The developmental origins of lung disease

Environmental exposures and lung disease

The role of epigenetics in the developmental origins of lung disease

Epigenetics and the programming of specific lung disease phenotypes

Conclusions

Chapter 9: Environmental contaminants and the immune system: A systems perspective

Abstract

An overview of the immune system

Immune disorders

Organic contaminant exposures and their links to immune dysfunction

Inorganic contaminants and their links to immune dysfunction

Conclusions

Chapter 10: Epigenetics, aging and early life

Abstract

Introduction to aging and epigenetics

Specific molecular epigenetic measures and their relationship with aging

Early-life DNA methylation aging

Conclusion

Section IV: Transgenerational epigenetic effects of the environment

Chapter 11: Intergenerational and transgenerational effects of environmental factors and a role for the epigenome

Abstract

Introduction

The developmental origins of health and disease

Intergenerational epigenetic inheritance

Transgenerational epigenetic inheritance

Conclusion

Section V: Epigenome-targeted therapies in environmental health science

Chapter 12: The role of nutrition and epigenetics in environmental toxicology

Abstract

Introduction

Interactions between inorganic contaminants and nutritional status

Interactions between organic contaminants and nutritional status

Future directions

Index

Copyright

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Contributors

Allison Aiello     Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Neil E. Alexis     Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Catherine M. Bulka     Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Vincenzo Cavalieri     Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, Palermo, Italy

Jeliyah Clark     Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Radhika Dhingra     Department of Environmental Sciences and Engineering, Institute for Environmental Health Solutions, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Lauren A. Eaves     Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Crisma Jazmin Emmanuel     School of Nursing, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Todd M. Everson

Department of Environmental Health

Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, United States

Rebecca C. Fry

Department of Environmental Sciences and Engineering

Institute for Environmental Health Solutions, Gillings School of Global Public Health

Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Amaree J. Gardner

Department of Environmental Sciences and Engineering

Institute for Environmental Health Solutions, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Akram Ghantous     Epigenetics Group, International Agency for Research on Cancer (IARC), Lyon, France

Zdenko Herceg     Epigenetics Group, International Agency for Research on Cancer (IARC), Lyon, France

Nicolette Jessen     School of Medicine, University of Utah, Salt Lake City, UT, United States

Lisa Joss-Moore     Department of Pediatrics, University of Utah, Salt Lake City, UT, United States

Blythe King     Department of Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Tracy A. Manuck     Department of Obstetrics and Gynecology, Institute for Environmental Health Solutions, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Carmen J. Marsit

Department of Environmental Health

Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, GA, United States

Alexei Novoloaca     Epigenetics Group, International Agency for Research on Cancer (IARC), Lyon, France

Jamaji C. Nwanaji-Enwerem     Medicine and Public Policy, Harvard Medical School & Harvard Kennedy School of Government, Boston, MA, United States

T. Michael O’Shea     Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Niharika Palakodety     Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Margaret Pinder     Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Julia E. Rager

Department of Environmental Sciences and Engineering

The Institute for Environmental Health Solutions, Gillings School of Global Public Health

Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Hudson P. Santos, Jr.

School of Nursing

Institute for Environmental Health Solutions, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Francie Sentilles     Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Alexandra Sexton-Oates     Epigenetics Group, International Agency for Research on Cancer (IARC), Lyon, France

Abhishek Venkatratnam     Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Cavin Ward-Caviness     Clinical Research Branch, U.S. Environmental Protection Agency, Chapel Hill, NC, United States

Preface

Rebecca C. Fry

In Environmental Epigenetics in Toxicology and Public Health, a transdisciplinary group of authors has come together to provide the reader with highlights of the scientific evidence of epigenetic processes linking exposure to environmental chemicals and health. A unique aspect of the book is the focus on translational research approaches that support relationships among exposure to environmental chemicals, the epigenome and health effects both early and later in life. Specifically, in many cases, the scientific evidence of chemically mediated disease is supported by both in vivo as well as human population-based research as highlighted throughout the book.

It is certainly not a new observation that chemicals in the environment can influence human health and disease. What is new, however, is the increasing understanding of the underlying biological mechanisms, obtained from in vivo, in vitro, and human population-based studies, suggesting that environmental chemicals can influence the complex epigenetic machinery. Highlighting developmental windows of susceptibility, there is also increasing evidence that both health effects evident early in life as well as later life health effects may be tied to such epigenetic modifications. It was a goal of this book to highlight a suite of complex environmental factors (both organic and inorganic in nature), their relationships with cancer and noncancer endpoints, and their established impact on various epigenetic components within the cell. The book provides evidence that environmental chemicals can cause dysregulation of CpG methylation, chromatin remodeling, and regulatory/noncoding RNAs. In several cases, these mechanisms by which epigenetic modifications occur are defined and the epigenetic modifications are also functionally tied to altered mRNA levels, protein levels, and disease outcomes.

In environmental health sciences and toxicology, it has become increasingly clear due to both targeted analysis as well as epigenome-wide association studies that exposure to environmental toxicants is tied to widespread and sometimes persistent alterations in the expression of messenger RNA. With importance for human health, these changes at the transcript level can be tied to protein expression changes that culminate in phenotypic differences at the organism level. With the exciting potential for a clinical opportunity for disease prevention and treatment, there is also the possibility that some epigenetic marks are potentially reversible. Thus, a more complete understanding of mechanisms of epigenetic regulation will aide in the development of new therapeutic options for the targeted removal of the potentially deleterious epigenetic alterations. If it is possible to modify disrupted epigenetic patterns via specific epigenome-targeted therapeutic interventions, it may be possible to prevent or reverse a variety of chronic diseases that are tied to environmental exposures to optimize health trajectories.

A summary of the chapters in the book is as follows: Chapter 1 provides a broad overview of epigenetics and its relevance in the context of the central dogma of biology. The field of epigenetics represents the study of the collection of potentially heritable changes in gene expression resulting from modifications above (epi-) the genome (genetics). Epigenetic mechanisms are responsive to intrinsic and environmental stimuli and collectively modify chromatin architecture and/or gene expression processing machinery, contributing to the regulation of gene expression without altering the underlying DNA nucleotide sequence. As critical determinants of gene expression levels and cellular function, understanding changes in epigenetic programming in response to chemical and nonchemical stressors continues to advance toxicology and environmental health research. Current insight into the biogenesis and roles of epigenetic modifications has been elucidated, in part, through scientific inquiry in a variety of model organisms. The chapter provides an overview of the literature surrounding three prominent epigenetic regulators: CpG methylation, chromatin remodeling, and regulatory/noncoding RNAs. In addition to describing the current understanding of the mechanisms underlying epigenetic control, the role of each modification in cellular processes is highlighted. The chapter also highlights questions that still remain surrounding tissue and temporal stability, transgenerational inheritance, and the replicability of these modifications.

Chapter 2 focuses on various epigenetic tools and technologies that can be used to assess epigenetic modifications and to characterize and quantify the transcriptome. The chapter covers the utilization of tools that assess epigenetic modifications that include high-throughput sequencing approaches, microarrays, and other specialized tools for studying mRNA, CpG methylation, chromatin structure and regulation, as well as miRNAs. Strategies for addressing common challenges of working with epigenetic data by covering statistical methods and pathway analyses. The chapter highlights challenges that remain in the lack of standardized analysis and processing protocols, degradation of genetic materials, and tissue variation.

Chapter 3 describes the use of various model organisms for studying the links between the environment, the epigenome, and disease. The chapter covers the utilization of a wide range of species spanning from unicellular eukaryotes to plants, fish, insects, and primates in experimental investigations. The use of the various model organisms has provided mechanistic causal data elucidating the role of environmental exposures on epigenetic modifications, maintenance and inheritance of that can potentially be translated to human applications. The chapter highlights the fact that as most studies to date have focused on DNA methylation, critical research gaps remain in relation to environmental modulation of histone posttranslational modifications, noncoding RNA, and chromatin structure.

Chapter 4 details the application of epidemiology within the context of environmental epigenetics. Epidemiology is the study of distributions and determinants of human disease within populations. Environmental epigenetics studies aim to identify and characterize intermediate biomarkers between exposures and outcomes, that can provide insights into delineating exposures, defining mechanisms of disease development, or highlighting disease risk. Such studies that focus on environmental health issues have exploded over the past two decades and have provided opportunities to consider a full spectrum of potential biomarkers as they relate to exposures and diseases. Thus, a proper understanding of epidemiologic principles within the context of epigenetic studies is critical to identifying useful biomarkers and drawing appropriate inferences. In this chapter, the common framework through which epigenetic epidemiology is conducted, key considerations in the design of epigenetic epidemiology studies, and approaches that are used to strengthen causal inferences are detailed.

Chapter 5 highlights that the etiology of adverse pregnancy and birth outcomes is multifactorial. In combination with known genetic factors, exposure to environmental contaminants has also been shown to be associated with adverse pregnancy and birth outcomes. In some cases, epigenetic mechanisms are believed to be critical in associations between environmental contaminant exposures and adverse pregnancy and birth outcomes. The chapter details studies that provide evidence relating exposures to both organic and inorganic contaminants to selected pathologies including spontaneous abortions and stillbirths, major structural anomalies, and pregnancy complications. Furthermore, the role of other environmental factors such as psychosocial stressors is considered as the maternal environment more broadly including experiences during pregnancy can strongly affect the fetal environment. Supporting data are drawn from both animal and human studies. Developmental windows of susceptibility and a role for the epigenome, where established, are emphasized.

Chapter 6 explores mechanisms by which exposure to environmental contaminants may cause irreversible disruption to brain structure and development. This chapter provides an overview of the neurodevelopmental effects of exposures to environmental contaminants. It covers a broad group of disorders, many of which are lifelong and have a large impact on individuals’ abilities to live independently. In addition, many are associated with high economic costs to the family and society at large. The chapter summarizes data from studies representing exposure to both organic contaminants and inorganic contaminants, including data from preclinical models and human populations. These studies provide information about developmental windows of vulnerability as well as genetic susceptibility to environmental factors. The chapter reviews accumulating evidence that early life inflammation contributes to adverse neurodevelopmental outcomes and evidence that environmental exposures can initiate fetal and neonatal inflammation. In addition, the chapter reviews epigenetic modifications that have the potential for linking environmental exposures to neurodevelopmental outcomes.

Chapter 7 discusses the involvement of epigenetic mechanisms, especially DNA methylation, in the link between early-life environmental exposures and cancer. Represented here is a growing pool of research evaluating factors such as pharmaceuticals, chemicals, infectious agents, socioeconomic and psychological factors in the development of cancer outcomes later in life. It is evident that exposure to environmental contaminants early in life has the potential to affect cancer development throughout the life course, however, future research is necessary to further develop our understanding of each contaminant's carcinogenic status, mechanistic association with cancer development, and the full extent to which epigenetic modifications are involved.

Chapter 8 evaluates epigenetics as a primary mechanism connecting early life exposure with later in life lung diseases under the developmental origins lung disease hypothesis, nested under the overarching developmental origins of health and disease hypothesis. This chapter investigates how exposure to environmental chemicals may impact the normal development and growth of the lung through reprogramming, resulting in altered lung structure, function, and ultimately disease. More specifically, this chapter investigates examples of how environmental exposures are a link between alterations in methylation, histone modifications, and noncoding RNA and neonatal lung development, asthma, chronic obstructive pulmonary disease, and adult lung diseases.

Chapter 9 explores epigenetic dysregulation as a mechanism for how environmental contaminants alter immune function. Immune system dysfunction has the potential to severely impact physiologic homeostasis which can increase the health risks associated with concomitant exposure to airborne pathogens (i.e., viral and bacterial infections) and other environmental stressors. This chapter introduces the main components of the immune system, autoimmune disorders, and how environmental contaminants including organic and inorganic pollutants alter immune function. It further discusses the association between environmental contaminants and immune diseases in both humans and mice and explores the role of the epigenome as an important link between exposure and disease. Organic contaminants including various pesticides, flame retardants, polychlorinated biphenyls, and per-fluorinated compounds substances have been linked to immune dysfunction in humans and the alteration of DNA methylation, chromatin remodeling, and ncRNA interactions. Inorganic contaminants such as inorganic arsenic and cadmium have also been linked to altered miRNA expression and DNA methylation.

Chapter 10 highlights the epigenetic aging as an emerging area of research with the potential to elucidate the life-long effects of early life exposures to chemicals. The chapter highlights epigenetic aging as the accumulation of select DNA methylation changes, that reflect a host of aging-related biological processes. Among the various markers of epigenetic aging, a large focus has been on particular sets of DNA methylation loci, termed epigenetic clocks. Several epigenetic clocks have demonstrated a robust relationship to chronological age across the lifespan. As such, we describe the current state of the literature examining the relationship of early life epigenetic aging (as estimated by epigenetic clocks) to exposure to chemical and nonchemical stressors and outcomes. Studies of early life epigenetic aging have focused on nonchemical stressors, such as the prenatal and maternal milieu, as the first modulating environment of the developing human, while the study of chemical exposures is thus far limited to inhaled pollutants. With respect to health outcomes, advanced epigenetic aging at birth has focused on the growth trajectory, while the impact of epigenetic aging in early life on long-term health outcomes remains understudied.

Chapter 11 highlights studies that examine the relationship of chemical exposure to health effects across multiple generations. Intergenerational epigenetic inheritance occurs when exposures in the parent generation (F0) impact the epigenome of the developing fetus (F1), or the offspring’s germ cells or the F2 generation to result in phenotypic alterations. Transgenerational epigenetic inheritance occurs when there is an impact on the epigenome that is evidenced in the F3 generation. Studies that focus on intergenerational inheritance are emphasized as there is generally more support in regard to the adaptive effects of exposures within shorter time periods (up to the F2 generation). Prevailing theories are discussed describing how exposure to environmental chemicals during prenatal periods of development are associated with later in life outcomes.

In Chapter 12, the role of nutrition is examined in the context of epigenetics in environmental toxicology. This chapter highlights the fact that nutritional status is one of the many determinants of health or disease with large implications for public health. In relation to toxicologic responses to chemical exposures, nutritional status can impact detoxifying mechanisms associated with chemical exposures subsequently influencing health effects. Several mechanisms by which nutrients impact responses to toxicants are discussed. A summary of the interaction between nutrition and DNA methylation in relation to toxicants in humans, rodents, and in vitro models is provided.

In summary, this book is meant for a wide range of readers who have an interest in understanding the role of the epigenome as a mediating factor linked toxic substances in the environment and human health and disease. It may be particularly interesting for those wishing to learn more about the current advances in epigenetics, environmental health, and toxicology research. It is the intention that this book will be helpful for the reader to have a deeper understanding of the epigenome as it relates to chemicals and disease.

Acknowledgments and Dedication

I would like to thank all of the authors for their hard work and dedication that was required for completion of this book: Allison Aiello, Neil E. Alexis, Catherine M. Bulka, Vincenzo Cavalieri, Jeliyah Clark, Radhika Dhingra, Lauren Eaves, Crisma Jazmin Emmanuel, Todd M. Everson, Amaree J. Gardner, Akram Ghantous, Zdenko Herceg, Nicolette Jessen, Blythe King, Lisa Joss-Moore, Tracy A. Manuck, Carmen J. Marsit, Jamaji C. Nwanaji-Enwerem, Alexei Novoloaca, T. Michael O’Shea, Niharika Palakodety, Margaret Pinder, Julia E. Rager, Hudson P. Santos, Jr., Francie Sentilles, Alexandra Sexton-Oates, Abhishek Venkatratnam, and Cavin Ward-Caviness. My special thanks to Celeste Carberry and Deb Bartle for their assistance with the book. This book simply would not have been possible without their collaboration and dedication. Finally, I would like to dedicate this book to Carol Remmer Angle, a pioneer in the field of toxicology and environmental health. Her persistence in the pursuit of scientific truth led her to climb over both physical and figurative barriers. Her determination and discoveries paved the way for generations to come.

Section I

Epigenetic mechanisms/machinery

Chapter 1: Epigenetics: An overview of CpG methylation, chromatin remodeling, and regulatory/noncoding RNAs

Jeliyah Clarka; Julia E. Ragera,b,c    a Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

b The Institute for Environmental Health Solutions, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

c Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

Abstract

The field of epigenetics represents the study of the collection of potentially heritable changes in gene expression resulting from modifications above (epi-) the genome (genetics). Epigenetic mechanisms are responsive to intrinsic and environmental stimuli and collectively modify chromatin architecture and/or gene expression processing machinery, contributing to the regulation of gene expression without altering the underlying DNA nucleotide sequence. As critical determinants of gene expression levels and cellular function, understanding changes in epigenetic programming in response to chemical and nonchemical stressors continues to advance toxicology and environmental health research. Current insight into the biogenesis and roles of epigenetic modifications has been elucidated, in part, through scientific inquiry in a variety of model organisms. The following chapter provides an overview of the literature surrounding three prominent epigenetic regulators: CpG methylation, chromatin remodeling, and regulatory/noncoding RNAs. In addition to describing current understanding of the mechanisms underlying epigenetic control, the role of each modification in cellular processes is highlighted.

Keywords

DNA methylation; Chromatin remodeling; Noncoding RNAs; Epigenetics; Human development

Introduction to epigenetics and its application to the central dogma of biology

Epigenetics represents a complex subfield of genetics, namely the collection of heritable modifications involved in regulating gene expression without altering the underlying DNA sequence [1]. To understand the role of epigenetics in establishing cellular function, it is imperative to highlight the overarching processes by which genes are expressed and where epigenetic modifications intervene. The central dogma of biology describes the flow of genetic information from DNA to RNA to proteins and occurs through two intermediate steps, namely transcription and translation (Fig. 1). During transcription, RNA is synthesized from the template strand of DNA by RNA polymerase through a series of steps appropriately named initiation, elongation, and termination. In most prokaryotes, a single RNA polymerase exists, while the complexity of eukaryotic genomes necessitates multiple species of RNA polymerases (I, II, and III) [2]. Within eukaryotes, genes encoding proteins are transcribed by RNA polymerase II. During initiation, RNA polymerase II binds the promoter region as part of a multiprotein-DNA complex with the aid of transcription factors [3]. Once bound, the polymerase moves along the helix in the 5′-to-3′ direction, unwinding the DNA to expose the template strand. During elongation, RNA polymerase II constructs a growing strand of ribonucleotides as it moves along the template in the 5′-to-3′ direction until encountering the terminator sequence, which signifies the end of transcription [2, 4]. Before translation commences, the nascent mRNA transcript must be modified through additional processing steps.

Fig. 1 The central dogma of biology. The central dogma describes the flow of genetic information from DNA → RNA → proteins and may be disrupted or modified by epigenetic mechanisms at any stage to regulate gene expression.

Further processing of the mRNA transcript within eukaryotes occurs through the addition of a cap at the 5′ end, splicing, and polyadenylation at the 3′ end [5]. Capping and polyadenylation protect the nascent mRNA transcript from degradation by phosphatases and nucleases [5, 6], while splicing excises viral sequences and introns to promote the ligation of exons [7, 8]. Alternative splicing may occur co- or posttranscriptionally and is hypothesized to be a source of diversity in the variety of structurally and functionally distinct mRNA, which can result in protein isoforms created from a single gene [9, 10]. Yet, the exact mechanisms of how and to what extent protein diversity is impacted by alternative splicing is debated and warrants further experimental evidence [10, 11]. After processing, the mature mRNA transcript exits the nucleus to be translated.

Translation begins in the cytoplasm of eukaryotes and is generally divided into three stages: initiation, elongation, and termination [12]. During initiation, the initiator methionyl transfer RNA (tRNA) and mature mRNA transcript bind to the small ribosomal subunit, which joins the large ribosomal unit to form a functional ribosome. Ribosomes contain three sites (peptidyl (P), aminoacyl (A), and exit (E)) in which tRNAs bind to produce polypeptide chains, and at the start of elongation the initiator methionyl tRNA is bound to the P site. With ribosome assembly complete, the mature mRNA transcript is translated into a chain of amino acids from nucleotide triplets during elongation.

The process of elongation has been described extensively elsewhere [12]. To summarize, tRNAs are escorted to the A site by an elongation factor to pair with the succeeding codon of the mRNA. As the peptide bond forms, the ribosome shifts three nucleotides along the mRNA strand, translocating the tRNA bound to the peptide chain to the P site as the tRNA formerly positioned in the P site moves to the E site before exiting [12]. This cycle forms a chain of amino acids in the 5′-to-3′ direction until the ribosome reaches a stop codon, and the ribosomal subunits and mRNA strand dissociate [12, 13]. Transcription and translation are highly regulated, though either process can be modified at any point to alter protein expression, and thus cellular phenotypes. Epigenetic mechanisms are important modifiers that hinder or promote the intermediate steps of the central dogma.

Every cell type in a multicellular organism contains identical genetic information, with very few exceptions. Cells differ in function and appearance according to their constituent proteins [14]. Thus, epigenetic mechanisms contribute to the establishment of cellular phenotypes by influencing patterns of gene expression that ultimately define the proteome, leading to unique cell structure and function. These mechanisms include CpG methylation, chromatin remodeling, and regulatory/noncoding RNAs (ncRNAs), with each influencing gene expression throughout multiple stages of the central dogma (Fig. 1). Epigenetic mechanisms collectively contribute to the regulation of gene expression and heredity and play critical roles in human development. In fact, the rapid development of different cell types during embryogenesis makes this a labile period in which the epigenetic modifications contributing to cell-type establishment can be disrupted. While highly susceptible to perturbations throughout the in utero period, alterations in epigenetic patterning can occur and accumulate across the life course of an organism, influencing gene expression patterns and disease susceptibility [15]. The relative plasticity of epigenetic modifications provides a model for studying the relationship between environmental exposures and human health and disease development, as epigenetic profiles correlate with chemical and social factors [16, 17]. Because of large intra- and intervariation between tissue types, ages, and populations, a referent epigenome for each cell type has been difficult to define [18]. This and other factors (e.g., limitations of epigenomic assays, temporal (in)stability) represent current challenges in determining whether epigenetic patterns might serve as biomarkers, mediators, or neutral by-products of environmental exposures [18].

In summary, much remains to be established concerning our understanding of epigenetic mechanisms. Still, the fields of environmental health and toxicology have been enriched from using epigenetics to understand the molecular mechanisms underlying the relationship between environmental exposures and human health outcomes. In this chapter, an overview of the biogenesis and various roles of CpG methylation, chromatin remodeling, and regulatory ncRNAs are discussed in the context of gene expression regulation and other cellular processes.

CpG methylation

The methylation landscape of a cell represents an important epigenetic regulator of gene expression that contributes to cellular processes mostly dependent on transcriptional silencing. CpG dinucleotides are genomic sites in which cytosines are adjacent and 5′ to guanines, with the p in CpG representing the DNA phosphate backbone. CpG methylation is an epigenetic modification describing the covalent transfer of a methyl group to the 5-carbon position of a CpG dinucleotide to form 5-methylcytosine (5mC) (Fig. 2) [19]. CpG methylation levels have been associated with a variety of factors, including age, cell type, disease status, pharmaceutical treatments, and environmental chemical exposures, and CpG methylation levels notably demonstrate varying degrees of temporal stability, in a genomic region-specific manner [20, 21]. Of note to the fields of environmental health and toxicology, some alterations in CpG methylation patterns persist throughout mitosis and in the absence of the conditions in which they were established [21]. The mechanisms and functions of CpG methylation have been investigated in the recent decades, and alongside the advent of biotechnological advances, it has become the most well-studied epigenetic mechanisms. Here, the regulation of and cellular processes driven by CpG methylation in the mammalian genome are reviewed.

Fig. 2 CpG methylation. The methylation of cytosine to 5-methylcytosine is mediated by DNA methyltransferases (DNMTs). This modification typically occurs at cytosines proximal to guanines in the linear sequence of nucleotides and acts as an important regulator of transcription.

The process of CpG methylation

During the process of CpG methylation, within mammalian cells, DNA methyltransferases (DNMTs) transfer methyl groups from S-adenosylmethionine, a universal methyl donor, to the fifth carbon of the cytosine ring to form 5-methylcytosine (5mC) [19]. DNMTs, along with ten-eleven translocation (TET) enzymes, are the most highlighted family of enzymes involved in the processes of CpG methylation. Together these enzymes contribute to the regulation of gene expression through establishing, maintaining, and erasing (demethylating) CpG methylation marks within cells [19, 22]. The CpG methylation landscape is governed by three overarching mechanisms, which include (1) the de novo establishment of methylation patterns (e.g., the establishment of methylation patterns in new cells early in development), (2) the maintenance of methylation patterns, and (3) the active removal of methyl groups (demethylation). To date, the properties destining genomic sequences for different mechanisms of CpG methylation (e.g., de novo, maintenance, and demethylation) at various developmental stages are not fully understood [23]. What has been identified are the family of enzymes and various proteins contributing to each process.

De novo establishment of CpG methylation patterns

De novo establishment refers to CpG methylation of DNA that has never been methylated, such as newly replicated daughter strands [22, 24]. The DNA methyltransferase 3 (DNMT3) family primarily carries out this process, though it is important to highlight that the functions of de novo and maintenance methylation are often shared across all groups of DNMTs [25, 26]. DNMT3A and DNMT3B collaborate to establish inaugural methylation patterns during the earliest stages of embryonic development. These methyltransferases have similar structures but are distinguished by their functions and gene expression patterns. DNMT3A is expressed ubiquitously and critical for the focal methylation of single-copy genes or regions undefined by long stretches of CpGs to be methylated [27]. In contrast, DNMT3B is not expressed in the majority of differentiated cells and preferentially targets highly repetitive, long stretches of CpGs (e.g., peri-centromeric regions consisting of repetitive genomic segments), suggesting it plays a more critical role during early development [19, 27]. In addition to these, DNMT3L is proposed to regulate de novo methylation patterns in early development, particularly in the methylation of imprinted and retrotransposon loci and X chromosome inactivation [28]. This enzyme shares homology with DNMT3A and DNMT3B and serves as a regulatory factor, but lacks methyltransferase activity [28, 29]. When coexpressed with DNMT3A, stimulation of DNMT3A by DNMT3L increases the fraction of methylated DNA molecules irrespective of the DNA sequence [28]. Thus, it is proposed to act as a general stimulatory factor for DNMT3A.

Maintenance of CpG methylation patterns

After DNA replication, the parent strand maintains CpG methylation patterns as the parent cell persists and functions within the body. The newly replicated CpGs on the daughter strand must also be methylated for the daughter cell to maintain the cellular phenotype and biological functions of the parent cell, hence the high expression levels of DNMT1 in all tissues [27]. Facilitated by the ubiquitin like with PHD and ring finger domains 1 (UHRF1) protein, DNMT1 preferentially binds these hemi-methylated strands at the replication fork to restore methylation patterns [30]. UHRF1 is a functional nuclear protein with a unique binding domain that recognizes hemi-methylated CpG sites [31, 32]. During the synthesis phase of the cell cycle, UHRF1 co-localizes with DNMT1, and has been shown to play an essential role in maintaining global and local CpG methylation patterns [31]. CpG methylation patterns are propagated with high fidelity and have the ability to remain stable for numerous cell generations, with experimental evidence attributing this to the recruitment of DNMT1 by UHRF1 [26, 32]. DNMT1 is also recruited at sites of DNA damage to restore methylation patterning during DNA repair [33] and is required for de novo methylation of non-CpG sites [30], as explained in the previous section. When DNMT1 fails to propagate methylation profiles onto nascent DNA strands, methyl groups are passively removed (demethylated), as in the case of epigenetic reprogramming when UHRF1 is transcriptionally silenced in the female germ line. Demethylation can also be mediated by TET enzymes in an active manner, independent of DNMT1 function [34].

Active demethylation of CpG dinucleotides

Active demethylation describes the process of 5mC erasure occurring through a series of enzyme-mediated reactions. As compared to passive demethylation, active demethylation is a more targeted form of 5mC erasure carried out by proteins in the TET enzyme family [34]. During active demethylation, 5mC is oxidized to unmodified cytosine via the thymine-DNA-glycosylase and DNA base excision repair (TDG/BER) pathway [34]. TET-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5-hmC) is the first enzymatic reaction to occur. Once 5-hmC is produced, TET enzymes proceed to convert 5-hmC to 5-formylcytosine (5-fmC) and 5-carboxylcytosine (5-caC). Each of the oxidized 5-substituents have unique steric and electronic properties and are found at varying levels in tissues, with 5-hmC being the most abundant [35, 36]. Emerging literature is identifying the importance of these additional forms of cytosine modification, with 5hmC implicated in the regulation of many cellular and developmental processes [37]. In a final step, TDG recognizes and excises the modified bases (5-fmC and 5-caC) [36]. This process yields an abasic site that undergoes base excision repair (BER), restoring the unmodified cytosine base.

CpG methylation as a regulator of gene expression

A role for CpG methylation in regulating gene expression has been elucidated through scientific inquiry, but the exact mechanisms by which CpG methylation represses or activates gene expression have yet to be fully resolved. In the context of CpG methylation in the promoter region of genes, it has been hypothesized that CpG methylation induces alterations of protein-DNA interactions [38], modulating the ability of transcription factors and other transcriptional machinery to access DNA. Some experimental evidence indicates that methyl moieties influence the binding of proteins to DNA, particularly those contributing to the organization of chromatin structure [38]. This model proposes that CpG methylation attracts proteins that bind methylated DNA, inhibiting the ability of other factors required for RNA synthesis to access the promoter region [38]. Proteins with methylated DNA-binding domains have been identified in complexes containing histone deacetylases, suggesting that CpG methylation and histone modifications may conspire to silence gene expression [39]. Additional parameters that have been associated with the degree of transcriptional silencing by CpG methylation include the density and location of methylated cytosines [39], with a general trend supporting that CpG methylation acts as a physical barrier to positive regulators of gene expression [27]. The methylation landscape of the human genome is reflective of this trend, with CpG methylation densely concentrated at transposable elements, owing to the evolutionary benefit of silencing jumping genes that induce mutations. Alternatively, CpG methylation of gene promoter regions is scant, with few exceptions, suggesting that these decreased methylation levels allow for the binding of transcription factors within promoter regions.

The distribution of CpG methylation influences the regulation of gene expression

Methylated cytosines are removed by enzymatic processes or spontaneous deamination, which forms thymine [27]. Unlike unmethylated cytosines, the transition mutations induced by spontaneous deamination of methylated cytosines are not readily recognized by DNA repair machinery. As a result, CpG dinucleotides have been steadily deaminated to thymine over the course of evolution, causing CpGs to account for less than 2% of dinucleotides in the entire genome [27]. While the degree of CpG methylation at the promoter region can be inversely associated with gene expression, experimental evidence has shown that other locations of CpG methylation also play a role in influencing whether genes will be transcribed. These include transposable elements, CpG islands, and gene body regions.

Transposable elements as indicators of global methylation patterns

Nearly one-third of the genome is comprised of transposable elements. These sequences or jumping genes move from one genomic site to another, altering genes or cis-regulatory elements by inducing mutations [40]. Due to the potential to propagate mutations in germ cells, transcriptional silencing of these regions is critical for maintaining genetic stability [41]. Two major classes of transposable elements exist (e.g., transposons and retrotransposons) and differ in the mechanisms by which they translocate. Short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs), both retrotransposons, have primarily been investigated in the context of CpG methylation and human health [41]. LINE-1 and Alu elements are examples frequently implemented in studies of global methylation patterns, owing to the fact that these repetitive sequences are highly conserved and ubiquitous in the human genome [42].

Silencing of LINE-1 and Alu elements is important to human health as aberrant transposable element activity is attributed to altered methylation profiles and may underly toxicity [41]. Some mechanisms by which transposable elements might be improperly silenced are related to the cell’s inability to methylate these genomic regions. Specifically, expression of transposable elements may occur in response to DNMT inhibition, the dysregulation of one-carbon metabolism, or via altered methylation persisting after DNA adduct repair [41]. Due to their ubiquity, methylation at repetitive elements has been implemented as a biomarker of global methylation patterns. Global hypomethylation, as indicated by decreased methylation at repetitive elements, has been associated with a variety of environmental exposures including: aflatoxin B1, air pollution, arsenic, and polycyclic aromatic hydrocarbons [43]. Global decreases in methylation are important to evaluate, as they can lead to overall genomic instability and increased frequency of mutations [44]. A second notable location of CpG sites is found in the context of CpG islands, genomic regions characterized by high CpG density.

CpG island promoters and gene-specific transcriptional regulation

Though CpG sites constitute a miniscule fraction of the genome (e.g., < 2%), CpG-rich regions, or CpG islands, are interspersed and associated with 70% of annotated gene promoters [45]. CpG islands are stretches of DNA (ranging from 300 to 3000 base pairs long) with a GC content greater than 50% and a ratio of CpG to GpC that is greater than 0.6 [27]. These regions are frequently unmethylated, even in the context of transcriptionally inactive genes, presumably owing to the benefits of a lower rate of mutations resulting from spontaneous deamination of methylated cytosines [19, 27, 45]. CpG islands are often associated with promoters in the mammalian genome, with approximately half containing transcription start sites [45].

There is an additional evidence supporting the role of CpG islands in promoter function, as a large class of CpG islands distal from annotated transcription start sites have shown evidence for promotion of gene transcription [46, 47]. These CpG island promoters are characterized by heterogeneous transcription start sites and transcription factor binding motifs [45]. While transcriptional silencing tends to precede CpG island methylation, studies have highlighted the stable transcriptional silencing of some genes via methylation of CpG islands [45]. In addition to CpG methylation, CpG islands are regulated by proteins influencing surrounding chromatin structure [45]. CpG island methylation is not tissue specific; however, the methylation of CpG island shores, regions immediately flanking and up to 2 kilobases away from CpG islands, is also correlated with gene expression and exhibits highly conserved patterns of tissue-specific methylation [19].

Alterations in CpG methylation associated with exposure to environmental toxicants

One valuable facet of epigenetic modifications is the ability to adapt and respond to environmental stimuli across the life course of an individual. This plasticity provides an excellent model for the study of environmentally induced changes in gene expression that may impact human health. Extant literature on the association between CpG methylation and various chemical and nonchemical exposures supports that CpG methylation patterning is perturbed by the environment. The direction of association and temporal stability of these changes varies by environmental factor, timing of the exposure, and genomic region under study.

As an example, in evaluating prenatal exposure to inorganic arsenic (iAs), we identified iAs-associated patterns of CpG methylation that corresponded to functional changes in mRNA expression levels [48]. iAs-associated CpG alterations were found to be both hypomethylated and hypermethylated and identified across multiple genomic regions, including: the first exon, untranslated regions, gene body, and upstream of the transcription start site [48]. In concordance with the role of CpG islands in regulating gene expression, many CpG islands were identified as those most significantly associated with gene expression levels, including those 200 base pairs upstream of the transcription start site. In utero and early-life periods represent critical windows of susceptibility in which the impact of environmental exposures are often amplified [18]. Still, these findings highlight the functional implications of alterations in CpG methylation associated with exposure to environmental toxicants that may lead to changes in gene expression and, ultimately, cellular phenotypes and human health.

Mechanisms underlying cellular processes

CpG methylation represents a dynamic tool for the tailoring of gene expression and underlies the regulation of various cellular processes. Specifically, due to its prominent roles in transcriptional repression and inducing heterochromatin structure, CpG methylation is central to processes involving differential, monoallelic, and/or repressed gene expression. Among these, genomic imprinting, epigenetic reprogramming, and cell differentiation are briefly outlined. For many biological processes in general, the identification of mechanisms by which they are established or maintained is still underway. A growing body of evidence has identified roles for epigenetic mechanisms, especially CpG methylation, in their function.

Genomic imprinting

Genomic imprinting is a mechanism by which genes are silenced or expressed in a parent-of-origin-dependent manner. Thus, imprinted genes represent those expressed monoallelically, according to the epigenetic marks established in the parental germ line. Imprinted genes are likely to have evolved as a mechanism for balancing parental resource allocation in offspring [49]. Two hallmark features of these genomic loci are that they (1) are protected from genome-wide demethylation in the early zygote and (2) are expressed in a parent-of-origin manner, with the mode of expression maintained throughout mitosis [38, 50, 51]. In concordance with their proposed evolutionary advantage, imprinted genes influence fetal and placental growth, as well as metabolism [51]. Notably, paternally expressed genes primarily represent potent growth enhancers, while most maternally expressed gene products contribute to growth restriction and the conservation of maternal resources [49, 50].

Genomic imprints are reprogrammed during gametogenesis, and in establishing parent-of-origin expression patterns, the homologous chromosomes must be distinguished [50]. The mechanisms by which maternal and paternal copies of imprinted genes become differentially silenced in the fertilized egg are unknown, but a role for CpG methylation in the establishment of imprinted genes is identified at imprinting control regions (ICRs). ICRs are cis-acting regulatory elements possessing differential epigenetic modifications, which are used to mediate or guide the establishment of parental allele-specific marks [52]. Most imprinted genes reside in approximately one million base pair long clusters containing ICRs [51]. These regions demonstrate differential methylation in gametes and during the preimplantation developmental phase, with most ICRs methylated on the maternal allele [51, 52]. DNMTs establish and contribute to the maintenance of methylation at ICRs, but it remains unclear exactly how ICRs are recognized by epigenetic machinery in the process of establishing genomic imprints [51].

Further emphasizing the role of methylation in imprinted gene function, developmental abnormalities associated with fetal growth and neurodevelopment have been identified in instances of aberrant imprinting methylation. Specifically, loss of monoallelic expression of imprinted genes results in disease (e.g., Prader-Willi syndrome and Angelman syndrome), with disease phenotypes also dependent on which parental allele is improperly silenced [50]. Even more notable, aberrant methylation of imprinted genes, in response to early-life environmental conditions, has been shown to persist throughout life and be associated with cardiometabolic disease and obesity, among other potential disease outcomes [53].

Epigenetic reprogramming

Epigenetic reprogramming describes the genome-wide erasure and reestablishment of epigenetic marks (e.g., CpG methylation) during gametogenesis and again postfertilization in the zygote [54, 55]. Epigenetic signatures maintain cell-specific transcriptional profiles and serve as a barrier to the fusion of highly differentiated gametes [54]. To allow for sexual reproduction and establish totipotency, epigenetic reprogramming occurs at two stages of development: during gametogenesis in primordial germ cells and postfertilization in the zygote [54]. The role of CpG methylation in epigenetic reprogramming has been studied throughout several species, and recent work is elucidating roles for collaboration between CpG methylation, histone modifications, and ncRNAs in reprogramming the early epigenetic landscape. The role of CpG methylation in epigenetic reprogramming has been the easiest to evaluate and is briefly described here in the context of mouse models, though there are subtle differences in the timing of events in humans [54].

After gastrulation, primordial germ cells within the epiblast proliferate and migrate to the future site of gonad development. Germ cells contain epigenetic marks (e.g., CpG methylation and histone modifications) derived from the embryonic cells they emerged from, so epigenetic reprogramming occurs during migration to ensure totipotency [54, 55]. The process includes separate waves of demethylation. During passive demethylation, in which de novo Dnmts and the key Dnmt1 cofactor (Uhrf1) are transcriptionally silenced, 5mC erasure occurs at promoters, gene bodies, and intergenic regions [54]. A second wave of active demethylation is required in primordial germ cells to reprogram imprinted methylation and key germ cell-related genes, though these exact mechanisms are still being elucidated [52, 54]. Of note to environmental toxicology and public health, the reestablishment of methylation marks occurs in a sex-specific manner, especially at imprinted genes, giving rise to sperm- and oocyte-specific methylation profiles [54, 55]. These differences are attributed to (1) sex-specific timing of germ cell-specific re-methylation and (2) cellular memory of parental alleles [54, 55]. As previously highlighted, once methylation at imprinted differentially methylated regions (DMRs) is established during gametogenesis, it is maintained and escapes the second epigenetic reprograming event.

The second major wave of epigenetic reprogramming occurs in the zygote within hours of fusion of the gametes. At this point, the zygote contains genomes contributed by each parent housed in separate pronuclei [55]. The reprogramming of the preimplantation embryo differs from that of the primordial germ cells in two primary ways: (1) demethylation kinetics differ for male and female-derived genomes and (2) methylation of imprinted loci is preserved, to propagate parent-of-origin methylation marks [55, 56]. The paternal genome is actively demethylated and acquires histone modifications, while reprogramming of the maternal epigenetic landscape is more static [56]. The passive demethylation of the maternal genome is attributed to the exclusion of DNMT1 from the pronucleus and protection from TET-mediated oxidation [55].

At most mammalian alleles, epigenetic reprogramming occurs in a predictable manner to promote fetal development. Though, as highlighted by the agouti mouse model, there are genomic loci in which reestablishment of epigenetic marks in early embryogenesis is probabilistic and influenced by the environment [57, 58]. A growing body of literature supports that epigenetic reprogramming of these loci can be influenced by maternal factors such as diet and exposure to environmental contaminants, influencing body size and