1. The Multifaceted Roles of YY1 in the Establishment of the Cellular Epigenetic Landscape

Raed Rizkallah

Yin Yang 1 (YY1) is an essential multifunctional protein needed for the regulation of a very wide spectrum of biological processes. Since its initial identification as a transcription factor that can repress, activate, or initiate transcription, YY1 has been shown to regulate hundreds of mammalian genes. Our knowledge of the mechanisms governing epigenetic regulation has grown exponentially over the past two decades. Mounting evidence in the scientific literature clearly indicates that YY1 is a central player in the establishment of the cellular epigenetic landscape. This chapter will focus on the multilayered roles of YY1 in epigenetic regulation with emphasis on the polycomb pathway, genomic imprinting, and X-chromosome inactivation. In addition, the known mechanisms of YY1 regulation will be discussed, along with possible implications on human health and disease through epigenetic disturbance.

2. SETting up the Epigenome Through the Histone Methyltransferase SETDB1

Zhuo Sun and Brian P. Chadwick

SETDB1 is unusual among the SET-domain containing histone methyltransferases (HMTases) due to the partitioning of its catalytic SET domain into two parts, hence its name "SET Domain Bifurcated 1". SETDB1 is one of several histone H3 lysine-9 HMTases, but is the only one that is essential in Drosophila. Through its ability to implement and maintain programmed silencing of genes that would otherwise interfere with differentiation and cell specialization, SETDB1 is indispensible for development from nematodes to mammals. In addition, SETDB1 is required to maintain pluripotency, in part through the silencing of endogenous retroviral elements that can act as promoters of chimeric transcripts or enhancers of surrounding genes when active. SETDB1 can be recruited to sites of action through a variety of proteins, among which targeting to KRAB-domain containing zinc finger proteins via the versatile adapter protein TRIpartite Motif-containing 28 (TRIM28) is best characterized. Through this large and diverse family of DNA binding proteins, TRIM28 guides SETDB1 to sites of action to shut down gene expression, which includes all silent alleles of known imprinted genes. SETDB1 is emerging as a key player in establishing and maintaining the epigenome. Due to this pivotal role, levels of SETDB1 protein needs to be strictly regulated to avoid it contributing to disease and cancer progression.

3. Sirtuin Deacetylases in Fungi: Connecting Metabolism to Lifecycle Progression, Stress Response, and Genome Stability

Laura N. Rusche, Ashleigh S. Hanner, Justin M.H. Heltzel, Kristen M. Humphrey, Shivali Kapoor and Christopher B. Rupert

Sirtuins are NAD⁺-dependent deacylases that regulate biological processes such as the maintenance of genome stability, response to stress, adaptation to low or poor nutrients, and initiation of life cycle events such as mating and sporulation. Because sirtuins require NAD⁺ for activity, changes in cellular metabolism that alter the NAD⁺/NADH ratio or NAD⁺ availability may influence the activity of sirtuins. Therefore, biological processes regulated by sirtuins could be linked to the metabolic state of the cell. Over the course of evolution, bringing new processes under the control of sirtuins could enable species to evolve novel responses to stresses that lower NAD+ levels. Here, we explore this hypothesis by examining how sirtuins contribute to the biology of a variety of fungal species.

4. Development-linked Differences in Cytosine 5-Hydroxymethylation in Mammalian DNA: Relationship to 5-Methylcytosine and Function

Melanie Ehrlich, Michelle Lacey, Guoqiang Zhang, Kenneth C. Ehrlich and Sriharsa Pradhan

There has been a burst of research activity since the recent definitive determination of 5-hydroxymethylcytosine (5hmC) as a differentiation-linked base in mammalian DNA. Differences in 5hmC tissue-specificity are even more striking than those for 5-methylcytosine (5mC), the DNA base from which 5hmC is derived. There are strong associations of 5hmC with extended promoter regions but its distribution around transcription start sites is different depending on the genes' transcriptional activity. The levels of 5hmC in the body of genes and in weak or strong enhancer-type chromatin partly correlate with transcription levels. We summarize some of the frequently used methods for analyzing 5hmC genomic profiles and recent evidence for correlations of genomic 5hmC with major histone modifications, differential splicing, differentiation, disease, and DNA demethylation. In addition, we compare DNA hydroxymethylation and DNA methylation in embryonic stem cells and the neural and skeletal muscle lineages as well as the distinct enrichment patterns of these two modifications along the genome. Because evidence indicates that 5hmC functions as both an intermediate in DNA demethylation and as a stable component of DNA with many different features from those of 5mC, 5hmC probably has a wide variety of roles in differentiation and sometimes in cell physiology.

5. The Identification of Mammalian Proteins Involved in Epigenetics

Luke Isbel, Harry Oey and Emma Whitelaw

Mutagenesis screening in Drosophila has identified genes involved in epigenetic processes and in most cases the mammalian homologues have been found to have similar functions. However, there are some gene silencing mechanisms that are present in mammals but absent in Drosophila, such as DNA methylation. So mutagenesis screens have also been carried out in the mouse and some novel genes have been found. Random mutagenesis screens in the mouse require a massive effort but they do facilitate the study of the phenotypic consequences of haploinsufficiency and this is providing us with clear associations between epigenetic dysfunction and human disease. In vitro RNAi screening in cell lines provides a high throughput alternative but faces some difficulties, in particular off- target effects. Biochemical approaches such as affinity pull down and mass spectrometry have provided researchers with a means to identify binding partners. Here we review the efforts made to identify the mammalian complexes necessary for epigenetic events. While the set of proteins found to be involved is now extensive, the challenge is to understand how they work together to achieve the highly regulated patterns of gene expression seen in the adult organism.

6. Chromatin-mediated Response to Stimuli

Daniel L. Vera, Lauren A. Cole, Benjamin Hoffman and Jonathan H. Dennis

In 1950, Edgar and Eileen Stedman hypothesized for the first time that "one of [histones'] physiological functions is to act as gene suppressors". This hypothesis implied that histones may block access to the underlying DNA sequence rendering it functionally unavailable. Sixty-four years later, the expectations of this statement remain unfulfilled. Indeed, cells with differing physiologies display unique gene expression patterns, specific DNA methylation patterns, and different histone post translational modifications. The scientific community had anticipated that cells with different physiologies would also have strikingly different nucleosome distributions; however this has not been the case. Cells with different physiologies have remarkably similar nucleosome distributions. Recently, it has been observed that nucleosome distributions do indeed change in a widespread manner in response to stimulus. This event had not been previously observed, as nucleosome redistributions are transient, returning to their basal positions later in the response. Additionally, a new set of nucleosome mapping experiments have led to the observation that nucleosomes exhibit differential sensitivity to nuclease. These results indicate that these fragile nucleosomes may be targets for regulatory factor binding. The discovery of the widespread but transient nature of nucleosome remodeling combined with the observation that basal nucleosome positions exhibit differential sensitivity to nuclease, has allowed for the development of a new model of a role for chromatin in genome regulation.

7. The Epigenetics of Centromere Function

Justyne E. Ross, Shannon M. McNulty and Beth A. Sullivan

The centromere is a complex chromosomal locus where the kinetochore is formed and microtubules attach during cell division. Centromere specification in eukaryotes largely depends on sequence-independent (epigenetic) mechanisms. In this chapter, we discuss current understanding of the epigenetic basis for centromere structure and function. In addition to endogenous centromeres in model organisms and humans, we review studies of atypical centromeres (neocentromeres, artificial chromosomes, dicentric chromosomes, engineered chromosomes) that have contributed to the current molecular understanding of centromere function. In particular, we focus on the behavior and regulation of the centromeric histone variant CENP-A, chromatin modifications, and transcription in centromere establishment and maintenance. Over the past three decades, our view of centromere biology has expanded significantly from the initial characterization of centromere proteins to a more mature understanding of the DNA, RNA, and protein components that constitute this complex chromosomal locus.

8. Dosage Compensation in Frogs and Toads

John H. Malone

Dosage compensation is a mechanism that facilitates changes in expression relative to DNA abundance. Intensive study of worms, flies, and human sex chromosomes during the last several decades has shown that intrinsic effects, noncoding RNA, macromolecular complexes, or a combination act to manipulate expression. The strength of studying these model organisms comes from detailed genetic knowledge and an array of molecular tools, however with the advent of whole genome sequencing, it becomes possible to understand dosage compensation in a wider evolutionary context. In this chapter, I provide an overview of dosage compensation and what is known about dosage compensation in frogs and toads. Frogs and toads represent the basal tetrapods and are useful models for understanding gene dosage during early development, a window that is difficult to study in vertebrates. While few studies exist, there are curious examples of dosage compensation in frogs. Applying genomic tools in frogs and toads will provide a better understanding for the role of compensation in solving problems of abnormal gene dosage, and create new opportunities for understanding the role of dosage compensation in evolution, genome function, and early development.

9. Ingenious Genes: The Diverse Roles of Long Noncoding RNA in Regulatory Processes

Emily M. Darrow and Brian P. Chadwick

The general principles of the Central Dogma describe the flow of genetic information from the transcription of DNA into RNA that is subsequently translated into protein. While there has long been an appreciation for those transcriptional units that do not complete this course and function at the level of RNA, the breadth and extent of molecules that fall into this category is undoubtedly far greater than could have been imagined in the early 1990's when the first long noncoding RNAs (lncRNA) were discovered. Experimental evidence firmly associates lncRNAs with epigenetic regulation of gene expression. In this chapter, we provide a broad overview of the many types and functions of lncRNA and briefly cover the various ways in which these RNA molecules impact and regulate protein-coding genes that do conform to the Central Dogma.

10. Epigenetic Mechanisms in Rett Syndrome

Janine M. LaSalle

Mutations in the X-linked gene MECP2 cause the neurodevelopmental disorder, Rett syndrome, an epigenetic disease at two levels. First, MECP2 is subject to X chromosome inactivation and MECP2 heterozygous female Rett patients are mosaics of both mutant and wild-type expressing cells. Differences in X chromosome inactivation patterns can therefore affect the phenotypic severity and pathogenesis of Rett syndrome and related disorders of MECP2 mutation or duplications in females. Second, MECP2 encodes methyl CpG binding protein 2, a member of the methyl binding domain (MBD) family of epigenetic readers of DNA methylation marks in chromatin. MeCP2 is an inherently disordered protein composed of different isoforms, post-translational modifications, and cofactor associations that dynamically change and mediate a variety of different functions in both neurons and non-neuronal tissues. Investigating both of these levels of epigenetic complexity of MECP2 is likely to be critical in understanding the molecular pathogenesis and disease progression, as well as developing effective therapies for Rett syndrome.

11. The Long and Short of Facioscapulohumeral Muscular Dystrophy

Sunny Das and Brian P. Chadwick

This chapter integrates research over the last few decades from various groups across the globe in order to provide a comprehensive account of different aspects of the debilitating muscle disorder Facioscapulohumeral muscular dystrophy (FSHD). FSHD is the third most common inherited form of muscular dystrophy, it is the first example of a macrosatellite-linked disease, displaying a complex pathogenesis due to interplay between genetic and epigenetic components. The chapter starts with the history of the disease and its clinical features. Various genetic and epigenetic determinants of the disease are subsequently detailed, aimed at understanding how they might be contributing to the phenotype. Finally, we discuss treatment of the disease and translational research for developing therapeutic approaches, along with associated concerns.

12. The Epigenetics of Nuclear Reprogramming to Pluripotency

Theodore P. Rasmussen

Recent exciting advances in cellular biology prove that it is possible to change virtually any cell type into another cell type. This is possible due to reprogramming, whereby the epigenome and transcriptome of cells is coaxed to assume the identity of another type of cell, often a pluripotent cell. In all cases, profound epigenetic changes are associated with successful reprogramming. This chapter discusses natural and artificial means of reprogramming, the chromatin-based mechanisms involved, and the problem of incomplete reprogramming and epigenetic memory. In particular, natural processes in which reprogramming is central are considered, including fertilization, preimplantation development, and reprogramming in the germ line. Artificially-induced reprogramming is also considered, to include somatic cell nuclear transfer (cloning), pluripotent stem cell derivation, fusion-mediated reprogramming, and the production of induced pluripotent stem cells. Many of these methods suffer from the problem of somatic cell epigenetic memory, and this is also considered.

13. Emerging Role of the Guanine-Quadruplex DNA Secondary Structure in Epigenetics

Aradhita Baral, Dhurjhoti Saha and Shantanu Chowdhury

Chromosomal DNA packaged as chromatin controls an intriguing variety of biological functions. Notably, a largely similar template of DNA sequence, comprising sequential arrangement of the four nucleic acid moieties, manages these functions. This is striking. In addition, this fraise an interesting question about whether the DNA sequence template plays a 'bystander' role in genome function or do locally attained structural formations also exert control. Recent studies directly implicate the chromatin in many processes including chromosome organization, transcription, replication and genomic stability. To some extent this is attributed to the supramolecular flexible structure of genomic DNA, which allows it to bend, twist and compress. Together these studies have pioneered a school of thought that DNA secondary structure derived from a particular encoded arrangement of DNA bases could participate in function. In this chapter we will focus on a particular type of DNA secondary structure - the G4 motif formed from a specific arrangement of guanine bases and discuss studies that together implicate a larger role of DNA structures in regulating genome-wide function.

14. Clinical Epigenetics in Cancer: Applications in Diagnosis, Prognosis and Therapy

María G. García, Estela G. Toraño, Agustín F. Fernández and Mario F. Fraga

Tumors harbor many epigenetic alterations affecting, among other things, genomic DNA methylation and histone post-translational modifications. Different epigenetic alterations are associated with the specific characteristics of each type of tumor, which has led to exploration of their potential use as clinical tumoral markers. Indeed, recent evidence increasingly points to epigenetic markers being a promising clinical tool for diagnosis, prognosis and therapy in cancer. Future challenges in the field include the routine use of genome-wide technologies and the development of new epigenetic drugs.

15. Environment and the Epigenetic Transgenerational Inheritance of Disease

Ingrid Sadler-Riggleman and Michael K. Skinner

Environmental factors can lead to transgenerational epigenetic changes in generations originating from ancestors exposed to environmental insults such as toxicants, abnormal nutrition or stress. These epigenetic germline changes can in turn increase disease susceptibility in offspring of exposed ancestors. The focus of this chapter is on environmentally induced epigenetic transgenerational inheritance of disease. The molecular mechanisms that allow these inherited epimutations to increase disease susceptibility will be reviewed. Environmental exposure specificity and exposure-specific disease development are also discussed.

16. Metabolic Inputs into Epigenetics

Scott J. Bultman

Gene-environment interactions converge at the level of the epigenome, which, in turn, influences transcriptome profiles and phenotypic outcomes. In this context, we must consider our diet as an amalgam of environmental factors that we are continuously exposed to and as something not restricted to caloric content. Certain bioactive food components and energy metabolites are known to modulate epigenomic marks and gene expression. In fact, many epigenetic enzymes require intermediary energy metabolites as essential co-factors. Collectively, this represents a suite of mechanisms that link nutrient availability with regulation of gene expression to maintain homeostasis. These mechanisms are important for human health, and perturbations can alter an individual's susceptibility to various disease states including cancer. This chapter discusses some of the better-understood relationships between diet/energy metabolites and DNA methylation, histone post-translational modifications, and ATPase chromatin-remodeling complexes in mammals. Each section includes specific examples of "metaboloepigenetics" that have been documented in humans and mouse models that are relevant to disease.

17. Environmental Exposures: Impact on the Epigenome

Jaclyn M. Goodrich and Dana C. Dolinoy

Exposures to environmental toxicants contribute significantly to the global burden of disease and are a prominent public health issue. Environmental toxicants exert their impact on health and disease in part by modifying the epigenome, DNA modifications that do not affect the underlying sequence but can result in altered gene expression and downstream phenotypic changes. While potentially reversible, toxicant-induced epigenetic change at vulnerable lifestages such as embryogenesis and adolescence may have a lasting effect on susceptibility to disease later in life and could even impact subsequent generations. In this chapter, we describe evidence for mitotically-inheritable epigenetic perturbation by toxicants from four chemical classes - heavy metals, persistent organic pollutants (POPs), endocrine disrupting chemicals (EDCs), and air pollution, a representative exposure mixture - from animal models and epidemiological cohorts. Research addressing mechanisms by which toxicants perturb the epigenome, the biological and functional significance of small epigenetic changes (e.g. DNA methylation, histone modifications, and non-coding RNA expression), and the impact of epigenetic change longitudinally on health outcomes are discussed. The translation of environmental epigenetics research to environmental policy and public health solutions will enhance chemical risk assessment and enable clinicians to identify at-risk populations prior to disease onset.