Epigenetics and Disease PDF

CHAPTER 6 Epigenetics and Disease...

CHAPTER 6 Epigenetics and Disease Diane P. Genereux http://evolve.elsevier.com/McCance/ Content Updates Chapter Summary Review Review Questions Case Studies Animations CHAPTER OUTLINE Overview of Epigenetic Mechanisms, 178 Detecting DNA Methylation in Single Molecules: Bisulfite DNA Methylation, 178 Conversion, 183 DNA Hydroxymethylation, 179 Detecting DNA Hydroxymethylation: Fluorescence Resonance Histone Modifications, 179 Energy Transfer, 183 Noncoding RNAs, 179 Identifying Histone-Modification States: Chromatin Epigenetics and Human Development, 180 Immunoprecipitation, 183 Genomic Imprinting, 180 Detecting DNA Accessibility States: DNase Hypersensitivity Prader-Willi and Angelman Syndromes, 180 Testing, 184 Beckwith-Wiedemann Syndrome, 181 Detecting DNA Accessibility States in Single Molecules: Assay for Russell-Silver Syndrome, 181 Transposase-Accessible Chromatin, 184 Epigenetics in Cognitive Development and Mental Health, 182 Publicly Available Resources Enable Exploration of Epigenetic Epigenetics and Ethanol Exposure in Utero, 182 States at Genome Scale, 184 Epigenetics and Mental Health, 182 Epigenetics and Cancer, 184 Fragile X Syndrome: A Genetic/Epigenetic Syndrome, 182 DNA Methylation and Cancer, 184 Exploring the Possible Multigenerational Persistence of Epigenetic Global Epigenomic Alterations and Cancer, 184 States That Arise in Response to Environmental Factors, 182 Epigenetic Screening for Cancer, 184 Epigenetics and Nutrition, 182 Misregulation of miRNAs in Cancer, 185 Epigenetics and Maternal Care, 183 Emerging Strategies for the Treatment of Epigenetic Disease, 185 Epigenetic Change over the Life Span, 183 DNA Demethylating Agents, 185 Twin Studies Reveal Epigenetic Changes over Time, 183 Histone Deacetylase Inhibitors, 185 Epigenetics and Aging, 183 miRNA Targeting, 186 Molecular and Computational Tools for Exploring Epigenetic Future Directions, 186 States, 183 Detecting DNA Methylation in Populations of Molecules: Methyl-Sensitive Restriction Enzymes, 183 177 178 UNIT II Genes, Gene-Environment Interaction, and Epigenetics Humans exhibit an impressive diversity of physical and behavioral traits. epigenetic perturbations are currently a focus of both preventative efforts Much of this diversity is attributable to genetic variation. Another and pharmaceutical interventions. substantial contributor is epigenetic (“upon genetic”) modification. The specific definition of epigenetic remains a topic of discussion among biologists.1,2 Under one definition, the term is reserved for modifications OVERVIEW OF EPIGENETIC MECHANISMS that are not encoded in nucleotide sequence but are nevertheless transmit- Epigenetic marks include DNA methylation and hydroxymethylation, ted when a somatic cell divides (mitotic inheritance), when gametes are chemical modifications that alter the charges of the histone proteins produced (germline inheritance), or both.3 In this chapter, the term around which DNA is wound for compaction within the nucleus, and epigenetic is used to refer to processes that modulate how a given set RNA-based mechanisms (Fig. 6.1). of genomic information gives rise to phenotype. Under this definition, epigenetic mechanisms include chemical modifications to DNA and DNA Methylation associated histones, the production of small regulatory RNA molecules, DNA methylation occurs through the attachment of a methyl group and, more generally, gene regulation by epigenetic processes at the level to the carbon-5 position of a cytosine. In the somatic cells of adults, of either transcription or translation. DNA methylation occurs principally at cytosines that are followed by Epigenetic modification is essential to fundamental processes of a guanine base (sometimes known as cytosines in “CpG dinucleotides”); human development, including the differentiation of embryonic stem in human embryonic stem cells, methylation also can occur at cytosines cells into specific cell types, and the inactivation of one of the two X outside of the CpG context.7 chromosomes in each cell of a genetic female. Some genes are said to DNA methylation plays a prominent role in human health and be imprinted, meaning that they are inherited from the mother and the disease. For example, in each cell of a normal human female, one of father in predictable and distinct epigenetic states. At imprinted loci, the two X chromosomes has dense methylation at most regions. Dense it is always either the allele from the mother or the allele from the father methylation is a key feature of heterochromatin, a structure consisting that is expressed in the offspring.4-6 of DNA tightly wound around histones into a condensed state and not A variety of diseases can result from abnormal epigenetic states. For actively transcribed. The heterochromatization of the so-called “inactive example, metabolic disease can occur when there is abnormal expression X” accounts for the cytologic observations that each cell from a human from both alleles at a locus that is typically imprinted; exposure to female has a region of densely stained DNA. This region was later environmental stressors can markedly increase the risk of abnormal confirmed to be the highly condensed inactive X chromosome.8,9 epigenetic states and is strongly associated with some cancers. Because Most regions of the other X chromosome in a cell from a human of their increasingly clear role in a wide range of pathologies, such female, the so-called active X chromosome, are largely devoid of DNA 3 RNA-based mechanisms 1 DNA methylation RNA strands Cytosine DNA methylation H1 H4 H3 H2B H2A 2 Histone post- translational modifications DNA Nucleosome Chromosome FIGURE 6.1 Three Major Types of Epigenetic Processes. Investigators are studying the following epigenetic mechanisms: (1) DNA methylation, (2) histone modifications, and (3) RNA based-mechanisms. See text for discussion.  CHAPTER 6 Epigenetics and Disease 179 methylation. The active X chromosome is said to be a transcriptionally and its associated histones and RNA molecules, is made up of individual active euchromatic state. Epigenetic inactivation of one of the two X nucleosome units. chromosomes occurs in each cell of a human female during gastrulation, When a given segment of DNA is bound tightly to its associated a phase of early embryonic development. The determination of which histones it is said to be heterochromatic, such that, as noted previ- chromosome is to be silenced, either the copy from the father or the ously in describing the inactive X chromosome, it is not accessible by copy from the mother, occurs at random and independently in each transcription factors and so cannot be used to guide the production of cell; the silent state of that chromosome and the active state of the other mRNA. By contrast, if a given segment of DNA is only loosely bound are inherited by descendant cells. If a woman’s two X chromosomes to associated histones it is said to be euchromatic, such that, as noted carry different alleles at a given locus, random X inactivation in early previously in describing the active X chromosome, transcription factors development can lead to somatic mosaicism, wherein differences between are able to access it and use it as a template for production of mRNA. the alleles active in two cells can confer two very different traits. Visually Whether a given segment of DNA is heterochromatic or euchromatic notable examples include the patchy coloration of calico cats and depends largely on chemical modifications to histone tails, parts of anhidrotic ectodermal dysplasia, the patchy presence and absence of sweat the histone-protein complex that extend away from the main part of glands in the skin of human females with one X chromosome bearing the nucleosome. a normal allele and one X chromosome bearing a mutant allele. It is Researchers are only beginning to understand the full diversity and important to note that somatic mosaicism because of random inactivation complexity of these histone modifications.17 The two modifications can arise for any X-encoded trait. As a result, females who inherit one best studied are histone acetylation and histone methylation. Histone normal allele and one disease allele at an X-encoded gene tend to have acetylation tends to diminish the positive charge of histones, reducing less severe disease phenotypes than do males whose lone X chromosome the strength of their binding to negatively charged DNA and, ultimately, bears a disease allele. This pattern is reflected in the typically lower making DNA more accessible for transcription. Histone methylation severity in females as compared to males for color blindness, fragile X can either increase or decrease the strength of bonding between DNA syndrome, and other phenotypes that arise from mutations on the X and histones, depending on the specific parts of the histones to which chromosome. the methyl groups are added. Aberrant DNA methylation, either the presence of dense methylation Whether individual segments of the genome are in heterochromatic where it is typically absent or the absence of methylation where it is or euchromatic states play a critical role in determining the developmental typically present, is associated with misregulation of tumor-suppressor potential of a given cell—that is, its capacity to give rise to a diverse genes and oncogenes. Specific alterations to DNA methylation states set of differentiated cell types. For example, chromatin states differ are a common feature of several human cancers, including those of the substantially between embryonic stem cells, which are poised to give colon10 and breast;11 the details of these alterations can be useful in rise to all of the different cell types that comprise an individual, and assessing disease prognosis12 (see Figs. 6.1 and 6.4; also see Chapter 12). terminally differentiated cells, which are committed to a specific developmental path. The fraction of DNA that is in the heterochromatic DNA Hydroxymethylation state typically increases as cells differentiate, in parallel to the reduction DNA hydroxymethylation is most common in cells that are undergo- in the number of genes that are active as a cell lineage transitions from ing epigenetic transition. DNA hydroxymethylation differs from DNA pluripotency, when they have the capacity to give rise to a large number methylation in that it is a hydroxymethyl group, rather than methyl of descendant cell types, to terminal differentiation, and when they are group, that is affixed to the C5 of cytosine. Discovery of this alternate able to produce only a cell of a single specified type.18 Mutations in DNA modification has helped to resolve long-standing uncertainties as genes that encode histone-modifying proteins have been implicated in to how genomic regions dense in DNA methylation undergo methylation various pathologic states, including congenital heart disease,19 highlighting loss, as observed, for example, in early embryonic development.13 histone modification as critical to normal development. In 2011, Wossidlo and colleagues14 found that genome-wide declines In contrast to the vast majority of other cell types, including oocytes, in DNA methylation in early murine zygotes occur in concert with sperm cells express not histones but protamines, which are evolutionarily genome-wide increases in DNA hydroxymethylation. They also found derived from histones.20 Protamines enable sperm DNA to achieve that this loss of methylation, as well as the parallel gain in hydroxy- compaction even greater than for the histone-bound DNA in somatic methylation, were obliterated in zygotes deficient in the Tet3 enzyme. cells. This tight compaction improves the hydrodynamic features of These two findings helped to establish that Tet enzymes can convert sperm cells, facilitating their movement. DNA methylation into hydroxymethylation. The name Tet derives from the term “ten-eleven translocation,” a reference to the discovery of these Noncoding RNAs enzymes through their association with a chromosomal translocation Noncoding RNAs (ncRNAs) play an important role in regulating a between chromosomes 10 and 11 that is commonly observed in some wide variety of cellular processes, including RNA splicing and DNA forms of leukemia.15 Though the specific functional impacts of hydroxy- replication. Of particular relevance to gene regulation are microRNAs methylation remain an area of active investigation, one recent proposal (miRNAs), which are encoded by DNA sequences approximately 22 is that it is associated with the activation of lineage-specific enhancers nucleotides long and typically reside within the introns of genes or in and so could play a role in specifying cell lineages.16 noncoding intergenic regions. In contrast to DNA methylation and histone modification, both of which principally impact gene expression Histone Modifications at the level of transcription, miRNAs typically modulate the stability Histones are the positively charged proteins around which negatively and translational efficiency of messenger RNAs (mRNAs) encoded at charged DNA molecules are wound, facilitating compaction of DNA other loci. Interaction between miRNAs and the mRNAs they target into the cellular nuclei. When all of the DNA that comprises the human for degradation is typically mediated by regions of partial sequence genome is wound around histones, it is only 1/40,000 as long as it complementarity. As a result, miRNAs can at once be specific enough would be in its uncondensed state. A set of histones and the segment so that they do not bind to all of the mRNAs in a cell and general of DNA wound around them are, together, known as individual enough to regulate a large set of different mRNA sequences. miRNAs nucleosomes. The total chromatin in a cell, a combination of DNA can also modulate translation by impairing ribosomal function. miRNAs 180 UNIT II Genes, Gene-Environment Interaction, and Epigenetics regulate diverse signaling pathways; those that stimulate cancer develop- meaning that the maternal copy is randomly chosen for inactivation ment and progression are called oncomirs. For example, miRNAs have in some somatic cells and the paternal copy is randomly chosen for been linked to carcinogenesis because they alter the activity of oncogenes inactivation in other somatic cells. For a third and smaller subset of and tumor-suppressor genes (see Chapter 12). Like other classes of autosomes (about 1%), either the maternal copy or the paternal copy genomic elements, the sequences that encode miRNAs can be tran- is imprinted, meaning that either the copy inherited through the sperm scriptionally silenced by DNA methylation. Insofar as the expression or the copy inherited through the egg is inactivated and remains in this of miRNAs can modulate the formation and growth of tumors, epigenetic inactive state in all of the somatic cells of the individual. modification of the sequences that encode them is a key area for The subset of genes that are subject to imprinting is highly enriched exploration of strategies for characterizing and perhaps even treating for loci relevant to organismal growth. The genetic conflict hypothesis22 cancer.21 was developed as a potential explanation for this pattern. The logic of this hypothesis is as follows. Although both the mother and the father benefit genetically from the birth and survival of offspring, their interests EPIGENETICS AND HUMAN DEVELOPMENT are not entirely aligned. Because mothers make a large physiologic Each of the cells in the early embryo has the potential to give rise to a investment in each child, it is in their evolutionary best interest to limit somatic cell of any type. These embryonic stem cells are therefore said the flow of energetic resources to any given offspring so as to maintain to be totipotent (“possessing all powers”). A key process in early their physiologic capacity to bear subsequent children. By contrast, development is the differential epigenetic modification of specific DNA except in cases of lifelong monogamy, it is in the best interest of the nucleotide sequences in these embryonic stem cells, ultimately leading fathers for their child to extract maximum resources from its mother, to the differential gene-expression profiles that characterize differentiated since the fathers’ reproductive success is tied to the survival of their somatic cell types. Early modifications ensure that specific genes are own child but not to the mother’s ability to bear additional offspring expressed only in the cells and tissue types in which their gene products in the future. Therefore, imprinted genes expressed from the maternal typically function (e.g., factor VIII expression primarily in hepatocytes, genome are predicted to limit offspring size, whereas imprinted genes and dopamine-receptor expression in neurons). expressed from the paternal genome are predicted to result in larger Epigenetic modifications early in development processes also highlight offspring. Available data are broadly consistent with these predictions. a fundamental feature of genetic as compared to epigenetic information: One hallmark of imprinting-associated disease is that the phenotype all of the cells in a given individual contain almost exactly the same of affected individuals is critically dependent on whether the mutation genetic information. It is the epigenetic information eventually placed is inherited from the mother or from the father. Some examples are “on top of ” these sequences that enables them to achieve the diverse included in the following. functions of differentiated somatic cells. A small percentage of genes, termed housekeeping genes, are necessary for the function and main- Prader-Willi and Angelman Syndromes tenance of all cells. These genes escape epigenetic silencing and remain A well-known disease example of imprinting is associated with a deletion transcriptionally active in all or nearly all cells. Housekeeping genes of about 4 million base pairs of the long arm of chromosome 15. When include those encoding histones, DNA and RNA polymerases, and this deletion is inherited from the father, the child manifests Prader-Willi ribosomal RNA genes. syndrome, with features that include short stature, hypotonia, small How do embryonic stem cells achieve epigenetic states typical of hands and feet, obesity, mild to moderate intellectual disability, and totipotency, whereby they can give rise to all of the diverse cell types hypogonadism23,24 (Fig. 6.2, A). The same deletion, when inherited from that comprise a fully developed organism? One explanation is that early the mother, causes Angelman syndrome, which is characterized by embryogenesis, which occurs during the 10 or so days just after fertiliza- severe intellectual disability, seizures, and an ataxic gait (Fig. 6.2, B).25 tion, is characterized by rapid fluctuation in genome-wide DNA These diseases are each observed in about 1 of every 15,000 live births; methylation densities. Fertilization triggers a global loss of DNA chromosome deletions are responsible for about 70% of cases of both methylation at most loci in both the oocyte-contributed and the sperm- diseases. The deletions that cause Prader-Willi and Angelman syndromes contributed genomes. This loss of methylation is accomplished in part are indistinguishable at the DNA sequence level and affect the same by suppression of the DNA methyltransferases, the enzymes that add group of genes. methyl groups to DNA. Methylation is not directly copied by the DNA It was unclear for several decades how the same deletion could replication process. Instead, immediately following replication, the produce such disparate results in different individuals. Further analysis methyltransferases read the pattern of methylation on the parent DNA showed that the 4 million base-pairs deletion (the critical region) contains strand and use that information to determine which daughter-strand several genes that are normally transcribed only on the copy of chromo- cytosines should be methylated. As embryonic cell division proceeds some 15 that is inherited from the father.26 These genes are imprinted in the absence of DNA methyltransferases, cell division continues, on the copy of chromosome 15 inherited from the mother, meaning eventually yielding cells that have nearly all of their loci in unmethylated, that they are transcriptionally silenced. Similarly, other genes in the transcriptionally active states. Around the time of implantation in the critical region are transcriptionally active only on the chromosome uterus, the DNA methyltransferases become active again, permitting copy inherited from the mother and are inactive, or imprinted, on the establishment of the cell lineage–specific marks required for the establish- chromosome inherited from the father (Fig. 6.3). If the single active ment of organ systems. copy of one of these genes is lost because of a chromosome deletion, then no gene product is produced, resulting in disease. Molecular analysis has revealed a great deal about genes in this GENOMIC IMPRINTING critical region of chromosome 15.26 The gene responsible for Angelman A baby inherits two copies of each autosomal gene: one from its mother syndrome encodes a ligase involved in protein degradation during brain and one from its father. For a large subset of these genes, expression is development, an observation that may help to explain the intellectual biallelic, meaning that both the maternally and the paternally inherited disability and ataxia observed in this disorder. In the brain, this gene copies contribute to offspring phenotype. As noted earlier, for another, is active only on the chromosome copy inherited from the mother. smaller subset of these genes, expression is stochastically monoallelic,22 Consequently, a maternally-transmitted deletion removes the single  CHAPTER 6 Epigenetics and Disease 181 active copy of this gene. Several genes in the critical region are associated with Prader-Willi syndrome and they are transcribed only on the Beckwith-Wiedemann Syndrome chromosome transmitted by the father. A paternally-transmitted deletion Another well-known example of imprinting is Beckwith-Wiedemann removes the only active copies of these genes producing the features syndrome, an overgrowth condition accompanied by an elevated risk of Prader-Willi syndrome. of cancer. Beckwith-Wiedemann syndrome is usually identifiable at birth because of the large size for gestational age, neonatal hypoglycemia, a large tongue, creases on the earlobe, and omphalocele (birth defect of infant’s intestines).27 Children with Beckwith-Wiedemann syndrome have an increased risk of developing Wilms tumor or hepatoblastoma. Both of these tumor types can be treated effectively if they are detected early; thus, screening at regular intervals is an important part of manage- ment. Some children with Beckwith-Wiedemann syndrome also develop asymmetrical overgrowth of a limb or one side of the face or trunk (hemihyperplasia). As with Angelman syndrome, a minority of Beckwith-Wiedemann syndrome cases (about 20% to 30%) are caused by the inheritance of two copies of chromosome 11 from the father and no copy of the chromosome from the mother, in a process known as uniparental disomy. Several genes on the short arm of chromosome 11 are imprinted on either the paternally- or the maternally-transmitted chromosome. These genes are found in two separate, differentially methylated regions (DMRs). In DMR1, the gene that encodes insulin-like growth factor 2 (IGF2) is inactive on the maternally-transmitted chromosome but active on the paternally-transmitted chromosome, such that a normal individual has only one active copy of IGF2. When two copies of the paternal chromosome are inherited (i.e., paternal uniparental disomy) or there is loss of imprinting on the maternal copy of IGF2, an active IGF2 gene is present in double dose. These changes produce increased levels of IGF2 during fetal development, contributing to the overgrowth features of Beckwith-Wiedemann syndrome. Thus, in contrast to Prader-Willi and Angelman syndromes, which are produced by a missing gene product, Beckwith-Wiedemann syndrome is caused, in part, by overexpression A B of a gene product. FIGURE 6.2 Prader-Willi and Angelman Syndromes. A, A child with Prader- Willi syndrome (truncal obesity, small hands and feet, inverted V-shaped upper Russell-Silver Syndrome lip). B, A child with Angelman syndrome (characteristic posture, ataxic gait, bouts Russell-Silver syndrome is characterized by growth retardation; pro- of uncontrolled laughter). (From Jorde LB, Carey JC, Bamshad MJ: Medical genetics, portionate short stature; leg length discrepancy; and a small, triangular- ed 4, Philadelphia, 2010, Mosby.) shaped face. About one-third of Russell-Silver syndrome cases are caused Deletion PWS region PWS region PWS region PWS region Deletion AS gene AS gene AS gene AS gene Chromosome 15 Chromosome 15 Chromosome 15 Chromosome 15 Active Inactive Prader-Willi syndrome Angelman syndrome FIGURE 6.3 Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS) Pedigrees. These pedigrees illustrate the inheritance patterns of Prader-Willi syndrome, which can be caused by a 4 million base pair deletion of chromosome 15q when inherited from the father. In contrast, Angelman syndrome can be caused by the same deletion but only when it is inherited from the mother. The reason for this difference is that different genes in this region are normally imprinted (inactivated) in the copies of 15q transmitted by the mother and the father in Prader-Willi and Angelman syndromes, respectively. (From Jorde LB, Carey JC, Bamshad MJ: Medical genetics, ed 4, Philadelphia, 2010, Mosby.) 182 UNIT II Genes, Gene-Environment Interaction, and Epigenetics by imprinting abnormalities of chromosome 11p15.5 that lead to X syndrome, characterized by reduced IQ and a set of behavioral down-regulation of IGF2 and therefore diminished growth. Another abnormalities. 10% of cases of Russell-Silver syndrome are caused by maternal uni- Remarkably, while possession of a large CG repeat in the FMR1 parental disomy. Thus, while it is upregulation, or extra copies, of active promoter dramatically increases the probability that an individual will IGF2 that causes overgrowth in Beckwith-Wiedemann syndrome, it is have fragile X syndrome, the disease can be at once present in males downregulation of IGF2 that causes the diminished growth seen in who have the large repeat and absent in their brothers who have inherited Russell-Silver syndrome. an allele of very similar size.40 This can be explained at least in part by the observation that acquisi- EPIGENETICS IN COGNITIVE DEVELOPMENT tion of methylation-based silencing at FMR1 is stochastic, meaning that the presence of a large repeat increases the probability of the dense AND MENTAL HEALTH promoter methylation that could lead to gene silencing, but does not Several lines of evidence suggest a role for epigenetic abnormalities in guarantee it. It remains to be seen whether dietary or environmental disorders of cognitive development. features can modulate the probability that dense methylation at FMR1 will accrue in individuals with the full-mutation allele. Epigenetics and Ethanol Exposure in Utero The impact of ethanol exposure in utero on skeletal and neural develop- ment was first reported in 1973,28 and led to broad awareness of fetal EXPLORING THE POSSIBLE alcohol syndrome. It was not until recently, however, that population- MULTIGENERATIONAL PERSISTENCE OF based and molecular-level studies began to clarify the epigenetic signals EPIGENETIC STATES THAT ARISE IN that are associated with these developmental abnormalities. Initially, researchers found that alcohol exposure in utero can impact the DNA RESPONSE TO ENVIRONMENTAL FACTORS methylation states of various genomic elements but without specific Emerging data suggest that conditions encountered in utero, during emphasis on loci directly relevant to skeletal and neural development.29 childhood, and even during adolescence or later can have long-term Later experiments revealed that treating cultured neural stem cells with impacts on epigenetic states. Some data have been interpreted to indicate ethanol impairs their ability to differentiate to functional neurons; this that epigenetic changes arising during an individual’s lifetime can be impairment seems to be correlated with aberrant, dense methylation transmitted through the germline, with the potential to impact gene at loci that are active in normal neuronal tissue.30 One possible explana- expression in future generations. tion for these effects is that ethanol exposure in utero modulates fetal Some reports of transgenerational inheritance of environment- expression of the DNA methyltransferases.31 induced epigenetics have failed to be replicated in subsequent work by other researchers. For example, Iqbal and colleagues41 sought to follow Epigenetics and Mental Health up on an earlier report42 that the epigenetic impacts of endocrine disrup- Several emerging lines of evidence suggest an association between altered tors can be transmitted, by an unknown mechanism, to the germline epigenetic states and mental health. A recent study found that children of the grandchildren of the mice exposed. Although they did confirm who grow up under conditions of poverty have atypical methylation at the capacity of endocrine disruptors to induce epigenetic changes in a serotonin receptor, suggesting DNA methylation as a mechanistic link tissues and gametes directly exposed, they found no evidence that any between early-life socioeconomic stress to an increased propensity to differences can persist to the germline of exposed animals’ grandchildren. depression.32 Similarly, in individuals with posttraumatic stress disorder Further investigation will be required to explain the disparity between (PTSD), alterations in gene expression in key neural pathways are these conflicting findings. A few examples of such apparent connections associated with atypical methylation in a large set of genes.33 Among between environment and inheritance of epigenetic states are discussed individuals with PTSD, specific changes in methylation are associated below; all of the examples remain under active investigation. with suicidal ideation and behavior.34 Autism-spectrum disorder also is associated with altered DNA methylation at some loci.35 In these Epigenetics and Nutrition cases, as in the connection of epigenetic abnormality to depression and During the winter of 1943, millions of people in urban areas of the PTSD, it is not clear whether epigenetic alteration plays a mechanistic Netherlands suffered starvation conditions as a result of a Nazi blockage role in the etiology of cognitive syndromes, or whether information that prevented shipments of food from agricultural area. When researchers on this link is useful principally for suggesting biomarkers potentially sought to investigate how exposure to famine during this Dutch Hunger useful for diagnosis. Winter had impacted individuals born in a historically prosperous country, they found that individuals who had been in utero during this Fragile X Syndrome: interval of severe nutritional deprivation were more likely to suffer A Genetic/Epigenetic Syndrome from obesity and diabetes as adults than were other individuals from Fragile X syndrome can be described as a genetic and epigenetic disease, the Netherlands. There also seemed to be a transgenerational impact, insofar as abnormalities of both sorts are observed at the fragile X locus, in that the children of individuals who were in utero during the Dutch FMR1, in affected individuals. The most common genetic abnormality Hunger Winter were found to be significantly smaller than were the at FMR1 involves expansion in the number of cytosine-guanine (CG) children of those not impacted by the blockade. Other datasets reveal dinucleotide repeats in the gene promoter. Females who have CG elevated risk of cardiovascular and metabolic disease for offspring of repeats in excess of the ⊕35 that are typical at this locus are at risk for individuals exposed during early development to fluctuations in food fragile X–associated primary ovarian insufficiency, characterized by an availability.43 elevated risk of early menopause.36 Males with moderate expansions are The specific molecular mechanisms that may mediate these apparent at risk of fragile X tremor ataxia syndrome (FXTAS), characterized by relationships between nutritional deprivation and disease risk on one a late-onset intention tremor.37 Both of these conditions seem to arise or more generations are largely unknown. From some animal models, through accumulation of excess levels of FMR1 mRNAs in nuclear it seems that the IGF2 gene is a possible target of epigenetic modifications inclusion bodies.38,39 Individuals with 200 repeats are at risk of fragile arising through nutritional deprivation. Exposure in utero and through  CHAPTER 6 Epigenetics and Disease 183 lactation to some chemicals (including bisphenol-A, a constituent of the impact of metformin on metabolism. However, metformin has plastics sometimes used in food preparation and storage) seems to lead recently been found specifically to alter the expression of SIRT1, the to epigenetic modifications similar to those that arise through nutritional gene encoding sirtuin, a protein that modulates the metabolic response deprivation in early life.44 to energy limitation. Therefore, it is possible that a drug that has long been used to treat insulin resistance may modulate epigenetic pathways, Epigenetics and Maternal Care with possible opportunities for life span extension in humans.49 There is increasing evidence that parenting style can impact epigenetic states, and that this information can be transmitted from one generation MOLECULAR AND COMPUTATIONAL TOOLS to the next. Mice and other rodents can exhibit two alternate styles of nursing behavior. One style is characterized by frequent arched-back FOR EXPLORING EPIGENETIC STATES nursing, with a high level of licking and grooming behavior. An alternate Epigenetic information is not encoded DNA sequence. Therefore, style is characterized by infrequent arched-back nursing and less licking specialized methods must be used to detect and quantify it. and grooming behavior. In one study,45 pups of mothers that engaged in frequent arched-backed nursing were found to have significantly Detecting DNA Methylation in Populations of lower methylation levels and higher transcription activity of a gluco- Molecules: Methyl-Sensitive Restriction Enzymes corticoid receptor–encoding locus. Because the glucocorticoid receptor Many restriction enzymes are sensitive to methylation, and so they is involved in a pathway that intensifies fearfulness and response to cleave DNA only at unmethylated cytosines. Clues to the overall level stress, these findings suggest that alteration to methylation states could of methylation in DNA isolated from a given individual can, therefore, help explain the finding that exposure to stress early in life can modulate be gathered by treating DNA with a methyl-sensitive enzyme, and then behavior in adulthood. These findings also highlight the concept that comparing banding patterns for enzyme-treated and nonenzyme-treated epigenetic processes can help store information about the environment, samples.53 and that the relevant epigenetic modifications can modulate behavior later in life. Detecting DNA Methylation in Single Molecules: Bisulfite Conversion EPIGENETIC CHANGE OVER THE LIFE SPAN Sometimes it is of interest to gather information about methylation on individual molecules. For example, among males who inherit a full- Twin Studies Reveal Epigenetic Changes mutation fragile X allele, there is a strong correlation between the severity over Time of the syndrome and the number of cells in which the fragile X allele Identical (monozygotic) twins, whose DNA sequences are essentially is densely methylated.40 To collect information on the methylation states the same, offer a unique opportunity to isolate and examine the trajectory of individual molecules, DNA can be subjected to bisulfite conversion of epigenetic change over the life span. A recent study found that, as before sequencing. Bisulfite treatment does not alter most nucleotides, twins age, they exhibit increasingly substantial differences in methylation including methylated cytosines, but deaminates unmethylated cytosines patterns of the DNA sequences of their somatic cells; these changes are to uracil.54 Because uracil complements adenine, not guanine, in double- often reflected in increasing numbers of phenotypic differences. Twins stranded DNA, methylated and unmethylated cytosines can be distin- with significant lifestyle differences (e.g., smoking vs. nonsmoking) guished in conventional sequence data, provided that the original tend to accumulate larger numbers of differences in their methylation sequence of that region is known and can be used to distinguish between patterns. These results, along with findings generated in animal studies, true thymines and converted, unmethylated cytosines. Either subcloning suggest that changes in epigenetic states may be an important part of followed by Sanger sequencing or single-molecule sequencing technolo- the aging process.46 gies can then be applied to ascertain the detailed methylation patterns of individual molecules. Epigenetics and Aging The occurrence of epigenetic change over the life span, as suggested by Detecting DNA Hydroxymethylation: studies in twins, is echoed by broader studies in both human and Fluorescence Resonance Energy Transfer nonhuman systems. In yeast, a shift in histone modification, as well as Following Wossidlo and colleagues’14 discovery that loss of DNA methyla- an overall decline in histone abundance, is a marker of replicative aging tion and the gain of DNA hydroxymethylation during early development, and is associated with dysregulation of gene expression.47 Chemical there has been considerable interest in exploring the specific functional labeling techniques have revealed that the genome-wide abundance of impacts and mechanisms of this transition. However, efforts in this hydroxymethylcytosine increases with time in the brains of adult mice.48 area have been hampered by lack of a method to detect relative levels In view of these and similar findings, some authors have proposed that of DNA methylation and DNA hydroxymethylation within individual senescence, itself, can be characterized as an epigenomic phenomenon.49 molecules. Recently, Song and colleagues55 introduced a promising From this perspective, while accrual of adverse exposures over the life solution. Under their new method, DNAs are first treated with two span is to some degree unavoidable, understanding environmental different fluorophores. Of these, one binds methyl groups and the other impacts on epigenetic states could lead to a new paradigm for life span binds hydroxymethyl groups. When samples are examined using fluo- extension. rescence assays, the methylation and hydroxymethylation can be dis- This apparent relationship between epigenetics and aging could help tinguished visually. This new method will undoubtedly enable critical to explain the long-standing observation that metformin, a drug used insight into the basic biology of epigenetic transitions and, perhaps, to treat insulin resistance, also is effective in slowing senescence in into the mechanisms of epigenetic disease. yeast.50 Studies in human populations suggest that long-term use of metformin may even extend mean life span in diabetic people beyond Identifying Histone-Modification States: that expected for untreated, nondiabetic individuals.51 Initially, these Chromatin Immunoprecipitation effects, which echo the increase in life span occurring under caloric The identity of DNA-bound proteins can provide important clues as restriction in primate models,52 were thought to operate directly through to the activity states of individual regions. The goal of chromatin 184 UNIT II Genes, Gene-Environment Interaction, and Epigenetics immunoprecipitation (ChIP) is to ascertain the sequences bound by Although it is possible for users to browse data using a visual interface, specific proteins of interest. Broadly, this is achieved in seven steps: the most efficient way to leverage these publicly available resources is 1. Cross-link DNA-bound proteins to the specific genomic regions to to use a command-line approach to download, process, and analyze which they are attached. At this step, binding is not specific, meaning available datasets. In just the past 3 years, data available through that all proteins, regardless of identity, are cross-linked to DNA. ROADMAP have provided insight into the genomic architecture underly- 2. Shear DNA, lysing DNA regions that are not protein bound. Proteins ing disorders of gonadal development,62 juvenile arthritis,63 and prevent sheering of the DNA to which they are bound. attention-deficit/hyperactivity disorder (HDAD).64 3. Select for proteins of interest, using a protein-specific antibody. 4. Capture protein-DNA complexes by using a secondary antibody EPIGENETICS AND CANCER that can bind to the first, and by washing away any remaining unbound DNA fragments. DNA Methylation and Cancer 5. Remove protein-DNA cross-links and wash away proteins. Some of the most extensive evidence for the association of epigenetic 6. Sequence DNA fragments to profile the portion of genome that modification with human disease comes from studies of cancer65,66 (Fig. was bound by the protein of interest. 6.4). Tumor cells often exhibit decreased methylation genome-wide, 7. Repeat to characterize DNAs bound to any other proteins of interest. relative to normal cells of the same type, which can increase the activity of oncogenes (see Chapter 12). Genome-wide loss of methylation tends Detecting DNA Accessibility States: to continue as tumors progress from benign neoplasms to malignancy. DNase Hypersensitivity Testing In addition, the promoter regions of tumor-suppressor genes are often DNase enzymes cleave uncondensed, transcriptionally active euchromatic hypermethylated, decreasing their rate of transcription and thus their DNA, but not condensed, transcriptionally silent heterochromatic DNA. ability to inhibit tumor formation. Hypermethylation of the promoter Genomic regions that are euchromatic in a given cell type therefore region of the RB1 gene is often seen in retinoblastoma,67 and hyper- can be identified through DNase treatment of bulk DNA samples. This methylation of the MLH1 locus is associated with colorectal cancer.68 approach can be readily applied at the genome scale,56 opening opportuni- It is important to note that the observation of perturbed DNA methyla- ties to compare transcriptionally active states across cells under various tion states in tumors is not, itself, an indication that perturbation in differentiation states and culture conditions. DNA methylation state is the proximate cause of cancer.69 However, as described in the following, the finding that many tumors have epigenetic Detecting DNA Accessibility States abnormalities hints at exciting new opportunities for minimally invasive in Single Molecules: Assay for cancer screening and diagnosis. Transposase-Accessible Chromatin Global Epigenomic Alterations and Cancer Like DNase hypersensitivity testing, the recently introduced assay for A major feature of one form of inherited colon cancer (hereditary transposase-accessible chromatin (ATAC-seq) is focused on identifying nonpolyposis colorectal cancer [HNPCC]) is the methylation of the genomic regions whose “open,” euchromatic states suggest that they promoter region of a gene, MLH1, whose protein product repairs could be transcriptionally active.57 However, in contrast to DNase assays damaged DNA. Inactivation of MLH1, a DNA mismatch repair under which transcriptionally active states are identified by their overall enzyme, is associated with accrual of DNA damage, another common sensitivity to DNase enzyme cleavage, ATAC-seq uses a transpose for feature of colon tumors (see Fig. 42.28). Abnormal methylation which cleavage of accessible sites is followed immediately by specific of tumor-suppressor genes also is common in the progression of ligation of a linker that bears barcodes that differ among cells. Genomic Barrett’s esophagus,70 a condition in which the esophagus is lined regions that have been ligated to adapters can then be isolated, ampli- with cells that have features typically associated with the lower fied, and sequenced. In contrast to earlier approaches, which collect intestinal tract. population-level summaries of active and inactive states of individual genomic locations, the use of single-cell barcodes in ATAC-seq enables Epigenetic Screening for Cancer comparison of epigenomes across individual cells in a sample.58 Using As noted previously, specific epigenetic abnormalities are often a defining single-cell ATAC-seq, one group found that there is an unexpectedly high feature of a tumor, but cannot necessarily be inferred to be the cause of level of variation among the epigenetic states of individual intestinal disease. Regardless of the nature of the connection between epigenetic lymphoid cells, and that this variation is shaped by the microbiome.59 abnormality and any particular cancer, the ability to screen for abnormal epigenetic states raises the possibility that epigenetic screening approaches Publicly Available Resources Enable Exploration could complement or even replace existing early-detection methods. of Epigenetic States at Genome Scale In some cases, epigenetic screening can be implemented using bodily One of the great advances of the genomics era is the emergence of fluids, such as urine or sputum, eliminating the need for the more public, online data repositories for researchers to share their findings, invasive, costly, and risky strategies that are currently standard. For enabling insights that might not be accessible from the work of any example, screening for epigenetic misregulation of miRNAs has shown single research group. Such publicly available datasets have provided promise as a tool for detecting and characterizing cancers of the colon,71 unprecedented opportunities to compare epigenomic profiles over breast,72 and prostate.73 Other epigenetics-based screening approaches developmental time and between tissues in disparate functional states. have shown promise for detecting and characterizing cancers of the The Encyclopedia of DNA Elements (ENCODE), a project supported bladder,74 lung,75,76 and prostate.77 by the National Human Genome Research Institute (NHGRI), is provid- Screening for epigenetic abnormality to detect cancer may be especially ing great insights as a catalog of the epigenomic signatures of cells powerful in conjunction with emerging methods for detecting tumors grown in culture.60 The Roadmap Epigenomics Project, a cooperative in blood and sputum samples.76,78 These “liquid-biopsy” methods are venture of epigenomic biologists worldwide sponsored by the U.S. minimally invasive and so could be useful not only for characterizing National Institutes of Health, is expanding this goal to provide informa- abnormalities after diagnosis but also for performing routine, simultane- tion on freshly sampled cells and tissues.61 ous screening for multiple types of cancers, even in individuals without  CHAPTER 6 Epigenetics and Disease 185 Fragile X Syndrome Fragile X syndrome results from mutational expansion at the FMR1 locus, followed by abnormal epigenetic inactivation. mRNA from FMR1 Normal FMR1 mutational expansion of repetitive region no mRNA dense methylation from FMR1 Fragile X CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 FMR1 A Facioscapulohumeral Muscular Dystrophy (FSHD) FSHD results from mutational contraction at the DUX4 locus, followed by abnormal epigenetic activation. no mRNA dense methylation from DUX4 Normal CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 DUX4 mutational contraction of repeated region mRNA little or no methylation from DUX4 FSHD DUX4 B FIGURE 6.4 Comparing the Molecular Mechanisms of Fragile X and FSHD. A, FMR1 in normal, expanded permutation, and full-mutation states. B, DUX4 in normal and contracted states. signs of disease, and for detecting the emergence of drug-resistance mutations in individuals undergoing chemotherapy. DNA Demethylating Agents 5-Azacytidine has been used as a therapeutic drug in the treatment Misregulation of miRNAs in Cancer of leukemia and myelodysplastic syndrome.79 A cytosine analog, 5- Hypermethylation also is seen in microRNA genes, which encode small azacytidine, is incorporated into DNA opposite its complementary (22 base pair) RNA molecules that bind to the ends of mRNAs, degrading nucleotide, guanine. Chemical differences between 5-azacytidine and them and preventing their translation. More than 1000 microRNA conventional cytosine (Fig. 6.5) cause it to achieve irreversible binding sequences have been identified in humans, and hypermethylation of to the DNA methyltransferases. As a result, the administration of specific subgroups of micoRNAs is associated with tumorigenesis. 5-azacytidine tends to reduce the density of DNA methylation,80 offering prospects for reversing the aberrant accumulation of methylation at EMERGING STRATEGIES FOR THE TREATMENT tumor-suppressor loci. Though associated with various side effects, including digestive disturbance, treatment with 5-azacytidine has shown OF EPIGENETIC DISEASE promise in the treatment of diseases, including pancreatic cancer81 and Epigenetic modifications are potentially reversible: DNA can be demethyl- myelodysplastic syndromes.82 ated, histones can be modified to change the transcriptional state of nearby DNA, and miRNA-encoding loci can be up- or down-regulated. Histone Deacetylase Inhibitors This raises the prospect for treating epigenetic disease with pharmaceuti- The activity of the histone deacetylases (HDACs) increases chromatin cal agents that directly reverse the changes associated with the disease compaction, decreasing transcriptional activity. In many cases, excessive phenotype. In recent years, interventions involving all three types of activity of HDACs results in transcriptional inactivation of tumor- epigenetic modulators (DNA methylation, histone modification, and suppressor genes, leading ultimately to the development of tumors. miRNAs) have shown considerable promise for the treatment of disease. Treatment with HDAC inhibitors, either alone or in combination with 186 UNIT II Genes, Gene-Environment Interaction, and Epigenetics NH2 NH2 other drugs, has shown promise in reducing cell-division rates in culture CH3 cells of cancers of the breast,83 prostate,84 and pancreas.85 N DNMT1+S-Adenosylmethionine N Most recently, HDACs have been used to treat some forms of leukemia and lymphoma that are characterized by transcriptional repression. N O N O In those diseases, HDACs become overly active, removing the acetyl H H groups that diminish the strength of bonds between DNA and histones, Cytosine 5-Methylcytosine ultimately preventing transcription factors from accessing DNA regions A whose activity is required for proper cellular function. A large number of drugs have proven useful in diminishing the removal of acetylation NH2 NH2 from histones. One drug in particular, abexinostat, is undergoing safety assessment to follow up on encouraging outcomes in a Phase N N N N II clinical trial.86 DNMT1+S-Adenosylmethionine N N O miRNA Targeting O H H A major challenge in developing drugs that modify epigenetic alterations 5-Azacytosine 5-Azacytosine is to target only the genes responsible for a specific cancer. Therapeutic B approaches that use microRNAs offer a potential solution to this problem because treatment can be targeted to individual loci that have aberrant FIGURE 6.5 5-Azacytosine as Demethylating Agent. A, Unmethylated cytosines expression using sequence characteristics of relevant RNA molecules. in DNA are typically subject to the addition of methyl groups by DNMT1, a DNA methyltransferase, using methyl groups supplied by the methyl donor S-adenosyl- methionine. B, In 5-Azacytosine, the 5′ carbon of cytosine is replaced with a nitrogen. FUTURE DIRECTIONS This chemical difference is sufficient both to block the addition of a methyl group Robust experimental observations are clarifying the roles of epigenetic and to confer irreversible binding to DNMT1. Incorporation of 5-azacytosine into states in determining cell fates and disease phenotypes. The well- DNA is therefore sufficient to drive passive loss of methylation from replicating documented involvement of epigenetic abnormalities in carcinogenesis DNA, and thus to reactivate hypermethylated loci. 5-Azacytosine, bound to a sugar, and the mounting evidence for these epigenetic changes in other common can be integrated into DNA, and has been administered with some success in diseases (discussed in other chapters) will likely elucidate possibilities treating epigenetic diseases that arise through hypermethylation of individual loci. for reversing the epigenetic abnormalities and possibly preventing their establishment in utero. SUMMARY REVIEW Overview of Epigenetic Mechanisms 8. The heritable transmission to future generations of epigenetic 1. Differences between the phenotypes of identical twins can provide modifications is called transgenerational inheritance. novel insights into epigenetic phenomena. 9. As twins age, they demonstrate increasing differences in methylation 2. To be both effective and safe, pharmaceutical strategies for treating patterns of their DNA sequences, causing increasing numbers of epigenetic abnormalities must be targeted to affected genomic phenotypic differences. regions. 10. In studies of twins with significant lifestyle differences (e.g., smoking 3. Epigenetics modification alters gene expression without changes vs. nonsmoking) large numbers of differences in their methylation to DNA sequence. patterns are observed to accrue over time. 4. Investigators are studying three major types of epigenetic processes: (a) DNA methylation, which results from attachment of a methyl Genomic Imprinting group to a cytosine; in the somatic cells, all or nearly all meth- 1. For some human genes, a given gene is transcriptionally active on ylation occurs at cytosines that are followed by guanines (“CpG only one copy of a chromosome (e.g., the copy inherited from the dinucleotides”); (b) histone modification, through the addition of father). On the other copy of the chromosome (the one inherited various chemical groups including methylation and acetylation; from the mother), the gene is transcriptionally inactive. This process and (c) noncoding RNAs (ncRNAs or miRNAs), short nucleotides of gene silencing, in which genes are silenced depending on which derived from introns of protein coding genes or transcribed as parent transmits them, is known as imprinting; the transcriptionally independent genes from regions of the genome whose functions, silenced genes are said to be “imprinted.” if any, remain poorly understood. MicroRNAs regulate diverse 2. When an allele is imprinted, it typically has dense DNA methylation; signaling pathways. the nonimprinted allele is typically not methylated. 5. DNA methylation is, at present, the best-studied epigenetic process. 3. A well-known disease example of imprinting is associated with a When a gene becomes heavily methylated, the DNA is less likely deletion of about 4 million base pairs (Mb) of the long arm of to be transcribed into mRNA. chromosome 15. When this deletion is inherited from the father, 6. Methylation, along with histone hypoacetylation and con- the child manifests Prader-Willi syndrome. densation of chromatin, inhibits the binding of proteins that 4. The same 4-Mb deletion, when inherited from the mother, causes promote transcription, such that the gene becomes transcriptionally Angelman syndrome. inactive. 5. Another well-known example of imprinting is Beckwith-Wiedemann 7. Environmental factors, such as diet and exposure to certain syndrome, an overgrowth condition accompanied by an increased chemicals, may cause epigenetic modifications. predisposition to cancer.  CHAPTER 6 Epigenetics and Disease 187 S U M M A R Y R E V I E W — cont’d 6. Whereas upregulation, or extra copies, of active IGF2 causes over- 2. Assay for transposase-accessible chromatin (ATAC-seq) uses growth in Beckwith-Wiedemann syndrome, downregulation of IGF2 a transposase to introduce DNA cell-specific barcodes into euchro- causes the diminished growth seen in Russell-Silver syndrome. matic DNA regions, permitting comparison of chromatin states across cells. Epigenetics in Cognitive Development and Mental Health 1. Consumption of alcohol during pregnancy has long been associated Epigenetics and Cancer with the cognitive abnormalities of fetal alcohol syndrome. Recent 1. The best evidence for epigenetic effects on disease risk comes from findings suggest that alcohol may affect these changes through altered studies of human cancer. methylation of genes involved in neuronal differentiation. 2. Methylation densities decline as tumors progress, which can increase 2. Individuals with autism and with PTSD have altered DNA methylation the activity of oncogenes, causing tumors to progress from benign profiles; current work is investigating the potential functional rel- neoplasms to malignancy. Additionally, the promoter regions of evance of these alterations. tumor-suppressor genes are often hypermethylated. These elevated 3. Fragile X syndrome results from a complex interaction of genetic methylation levels decrease their rate of transcription at these critical and epigenetic abnormalities. genes, thus reducing the ability to inhibit tumor formation. 4. Fetal alcohol syndrome, which results from ethanol exposure in 3. Hypermethylation also is seen in microRNA genes and is associated utero, may be mediated by the repressive impact of ethanol on the with tumorigenesis. DNA methyltransferases. 4. 5-Azacytidine, a demethylating agent, has been used as a therapeutic 5. Both abnormal gain of methylation, as in the case of fragile X drug in the treatment of leukemia and myelodysplastic syndrome. syndrome, and abnormal loss of methylation, as in the case of FSHD, can produce disease phenotypes. Emerging Strategies for the Treatment of Epigenetic Disease 1. In diseases associated with aberrant, dense methylation of individual Exploring the Possible Multigenerational Persistence loci, treatment with 5-azacytidine, a cytosine analog that is refractory of Epigenetic States That Arise in Response to to the addition of methyl groups, can lead to clinically beneficial Environmental Factors reduction in methylation densities. 1. Events encountered in utero, in childhood, and in adolescence can 2. miRNAs can be used to regulate the expression of loci whose altered result in specific epigenetic changes that yield a wide range of epigenetic states are associated with disease. phenotypic abnormalities, including metabolic syndromes. 3. Histone–deacetylase inhibitors have shown promise in treating cancers of the breast, prostate, and pancreas. Epigenetic Change over the Life Span 1. Identical twins diverge in their epigenetic states over time at Future Directions rates potentially modulated by environmental factors, such as 1. Robust experimental observations are defining the roles of epigenetic tobacco use. states in shaping cell fates. 2. Metformin, a drug useful in treating insulin resistance, also may 2. The well-documented involvement of epigenetic abnormalities in prolong life through epigenetic mechanisms. carcinogenesis and the mounting evidence for these epigenetic changes in other common diseases (discussed throughout the text) will likely Molecular and Computational Tools for elucidate new therapies with the possibilities of reversing the epi- Exploring Epigenetic States genetic abnormalities. 1. Bisulfite conversion induces chemical changes in the binding proper- ties of cytosine and methylcytosine, such that they can be distinguished in sequence data. KEY TERMS Angelman syndrome, 180 Euchromatin (euchromatic), 179 Oncomir, 180 Assay for transposase-accessible chromatin Fragile X syndrome, 182 Prader-Willi syndrome, 180 (ATAC-seq), 184 Genetic conflict hypothesis, 180 Protamines, 179 Beckwith-Wiedemann syndrome, 181 Heterochromatin (heterochromatic), 178, 179 Roadmap Epigenomics Project, 184 Biallelic, 180 Histone, 179 Russell-Silver syndrome, 181 Bisulfite conversion, 183 Histone modification, 179 Somatic mosaicism, 179 Chromatin immunoprecipitation (ChIP), 183 Housekeeping gene, 180 Totipotent, 180 DNA hydroxymethylation, 179 Imprinted, 180 Transcription factor, 179 DNA methylation, 178 MicroRNA (miRNA), 179 Tumor-suppressor gene, 179 DNA methyltransferase, 180 Monoallelic, 180 Uniparental disomy, 181 Embryonic stem cell, 180 Noncoding RNA (ncRNA), 179 X inactivation, 179 Encyclopedia of DNA Elements (ENCODE), 184 Nucleosome, 179 Epigenetic, 182 Oncogene, 179