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DNA stands for Deoxyribonucleic Acid, and although each persons DNA is unique to that individual, it is 99.9% similar to every other human on the planet. [Image: CC0 Public Domain, https://goo.gl/m25gce%5D

Early childhood is not only a period of physical growth; it is also a time of mental development related to changes in the anatomy, physiology, and chemistry of the nervous system that influence mental health throughout life. Cognitive abilities associated with learning and memory, reasoning, problem solving, and developing relationships continue to emerge during childhood. Brain development is more rapid during this critical or sensitive period than at any other, with more than 700 neural connections created each second. Herein, complex geneenvironment interactions (or genotypeenvironment interactions, GE) serve to increase the number of possible contacts between neurons, as they hone their adult synaptic properties and excitability. Many weak connections form to different neuronal targets; subsequently, they undergo remodeling in which most connections vanish and a few stable connections remain. These structural changes (or plasticity) may be crucial for the development of mature neural networks that support emotional, cognitive, and social behavior. The generation of different morphology, physiology, and behavioral outcomes from a single genome in response to changes in the environment forms the basis for phenotypic plasticity, which is fundamental to the way organisms cope with environmental variation, navigate the present world, and solve future problems.

The challenge for psychology has been to integrate findings from genetics and environmental (social, biological, chemical) factors, including the quality of infantmother attachments, into the study of personality and our understanding of the emergence of mental illness. These studies have demonstrated that common DNA sequence variation and rare mutations account for only a small fraction (1%2%) of the total risk for inheritance of personality traits and mental disorders (Dick, Riley, & Kendler, 2010; Gershon, Alliey-Rodriguez, & Liu, 2011). Additionally, studies that have attempted to examine the mechanisms and conditions under which DNA sequence variation influences brain development and function have been confounded by complex cause-and-effect relationships (Petronis, 2010). The large unaccounted heritability of personality traits and mental health suggests that additional molecular and cellular mechanisms are involved.

Epigenetics has the potential to provide answers to these important questions and refers to the transmission of phenotype in terms of gene expression in the absence of changes in DNA sequencehence the name epi- (Greek: - over, above) genetics (Waddington, 1942; Wolffe & Matzke, 1999). The advent of high-throughput techniques such as sequencing-based approaches to study the distributions of regulators of gene expression throughout the genome led to the collective description of the epigenome. In contrast to the genome sequence, which is static and the same in almost all cells, the epigenome is highly dynamic, differing among cell types, tissues, and brain regions (Gregg et al., 2010). Recent studies have provided insights into epigenetic regulation of developmental pathways in response to a range of external environmental factors (Dolinoy, Weidman, & Jirtle, 2007). These environmental factors during early childhood and adolescence can cause changes in expression of genes conferring risk of mental health and chronic physical conditions. Thus, the examination of geneticepigeneticenvironment interactions from a developmental perspective may determine the nature of gene misregulation in psychological disorders.

This module will provide an overview of the main components of the epigenome and review themes in recent epigenetic research that have relevance for psychology, to form the biological basis for the interplay between environmental signals and the genome in the regulation of individual differences in physiology, emotion, cognition, and behavior.

Almost all the cells in our body are genetically identical, yet our body generates many different cell types, organized into different tissues and organs, and expresses different proteins. Within each type of mammalian cell, about 2 meters of genomic DNA is divided into nuclear chromosomes. Yet the nucleus of a human cell, which contains the chromosomes, is only about 2 m in diameter. To achieve this 1,000,000-fold compaction, DNA is wrapped around a group of 8 proteins called histones. This combination of DNA and histone proteins forms a special structure called a nucleosome, the basic unit of chromatin, which represents a structural solution for maintaining and accessing the tightly compacted genome. These factors alter the likelihood that a gene will be expressed or silenced. Cellular functions such as gene expression, DNA replication, and the generation of specific cell types are therefore influenced by distinct patterns of chromatin structure, involving covalent modification of both histones (Kadonaga, 1998) and DNA (Razin, 1998).

Importantly, epigenetic variation also emerges across the lifespan. For example, although identical twins share a common genotype and are genetically identical and epigenetically similar when they are young, as they age they become more dissimilar in their epigenetic patterns and often display behavioral, personality, or even physical differences, and have different risk levels for serious illness. Thus, understanding the structure of the nucleosome is key to understanding the precise and stable control of gene expression and regulation, providing a molecular interface between genes and environmentally induced changes in cellular activity.

DNA methylation is the best-understood epigenetic modification influencing gene expression. DNA is composed of four types of naturally occurring nitrogenous bases: adenine (A), thymine (T), guanine (G), and cytosine (C). In mammalian genomes, DNA methylation occurs primarily at cytosine residues in the context of cytosines that are followed by guanines (CpG dinucleotides), to form 5-methylcytosine in a cell-specific pattern (Goll & Bestor, 2005; Law & Jacobsen, 2010; Suzuki & Bird, 2008). The enzymes that perform DNA methylation are called DNA methyltransferases (DNMTs), which catalyze the transfer of a methyl group to the cytosine (Adams, McKay, Craig, & Burdon, 1979). These enzymes are all expressed in the central nervous system and are dynamically regulated during development (Feng, Chang, Li, & Fan, 2005; Goto et al., 1994). The effect of DNA methylation on gene function varies depending on the period of development during which the methylation occurs and location of the methylated cytosine. Methylation of DNA in gene regulatory regions (promoter and enhancer regions) usually results in gene silencing and reduced gene expression (Ooi, ODonnell, & Bestor, 2009; Suzuki & Bird, 2008; Sutter and Doerfler, 1980; Vardimon et al., 1982). This is a powerful regulatory mechanism that ensures that genes are expressed only when needed. Thus DNA methylation may broadly impact human brain development, and age-related misregulation of DNA methylation is associated with the molecular pathogenesis of neurodevelopmental disorders.

The modification of histone proteins comprises an important epigenetic mark related to gene expression. One of the most thoroughly studied modifications is histone acetylation, which is associated with gene activation and increased gene expression (Wade, Pruss, & Wolffe, 1997). Acetylation on histone tails is mediated by the opposing enzymatic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Kuo & Allis, 1998). For example, acetylation of histone in gene regulatory regions by HAT enzymes is generally associated with DNA demethylation, gene activation, and increased gene expression (Hong, Schroth, Matthews, Yau, & Bradbury, 1993; Sealy & Chalkley, 1978). On the other hand, removal of the acetyl group (deacetylation) by HDAC enzymes is generally associated with DNA methylation, gene silencing, and decreased gene expression (Davie & Chadee, 1998). The relationship between patterns of histone modifications and gene activity provides evidence for the existence of a histone code for determining cell-specific gene expression programs (Jenuwein & Allis, 2001). Interestingly, recent research using animal models has demonstrated that histone modifications and DNA methylation of certain genes mediates the long-term behavioral effects of the level of care experienced during infancy.

The development of an individual is an active process of adaptation that occurs within a social and economic context. For example, the closeness or degree of positive attachment of the parent (typically mother)infant bond and parental investment (including nutrient supply provided by the parent) that define early childhood experience also program the development of individual differences in stress responses in the brain, which then affect memory, attention, and emotion. In terms of evolution, this process provides the offspring with the ability to physiologically adjust gene expression profiles contributing to the organization and function of neural circuits and molecular pathways that support (1) biological defensive systems for survival (e.g., stress resilience), (2) reproductive success to promote establishment and persistence in the present environment, and (3) adequate parenting in the next generation (Bradshaw, 1965).

The most comprehensive study to date of variations in parental investment and epigenetic inheritance in mammals is that of the maternally transmitted responses to stress in rats. In rat pups, maternal nurturing (licking and grooming) during the first week of life is associated with long-term programming of individual differences in stress responsiveness, emotionality, cognitive performance, and reproductive behavior (Caldji et al., 1998; Francis, Diorio, Liu, & Meaney, 1999; Liu et al., 1997; Myers, Brunelli, Shair, Squire, & Hofer, 1989; Stern, 1997). In adulthood, the offspring of mothers that exhibit increased levels of pup licking and grooming over the first week of life show increased expression of the glucocorticoid receptor in the hippocampus (a brain structure associated with stress responsivity as well as learning and memory) and a lower hormonal response to stress compared with adult animals reared by low licking and grooming mothers (Francis et al., 1999; Liu et al., 1997). Moreover, rat pups that received low levels of maternal licking and grooming during the first week of life showed decreased histone acetylation and increased DNA methylation of a neuron-specific promoter of the glucocorticoid receptor gene (Weaver et al., 2004). The expression of this gene is then reduced, the number of glucocorticoid receptors in the brain is decreased, and the animals show a higher hormonal response to stress throughout their life. The effects of maternal care on stress hormone responses and behaviour in the offspring can be eliminated in adulthood by pharmacological treatment (HDAC inhibitor trichostatin A, TSA) or dietary amino acid supplementation (methyl donor L-methionine), treatments that influence histone acetylation, DNA methylation, and expression of the glucocorticoid receptor gene (Weaver et al., 2004; Weaver et al., 2005). This series of experiments shows that histone acetylation and DNA methylation of the glucocorticoid receptor gene promoter is a necessary link in the process leading to the long-term physiological and behavioral sequelae of poor maternal care. This points to a possible molecular target for treatments that may reverse or ameliorate the traces of childhood maltreatment.

Several studies have attempted to determine to what extent the findings from model animals are transferable to humans. Examination of post-mortem brain tissue from healthy human subjects found that the human equivalent of the glucocorticoid receptor gene promoter (NR3C1 exon 1F promoter) is also unique to the individual (Turner, Pelascini, Macedo, & Muller, 2008). A similar study examining newborns showed that methylation of the glucocorticoid receptor gene promoter maybe an early epigenetic marker of maternal mood and risk of increased hormonal responses to stress in infants 3 months of age (Oberlander et al., 2008). Although further studies are required to examine the functional consequence of this DNA methylation, these findings are consistent with our studies in the neonate and adult offspring of low licking and grooming mothers that show increased DNA methylation of the promoter of the glucocorticoid receptor gene, decreased glucocorticoid receptor gene expression, and increased hormonal responses to stress (Weaver et al., 2004). Examination of brain tissue from suicide victims found that the human glucocorticoid receptor gene promoter is also more methylated in the brains of individuals who had experienced maltreatment during childhood (McGowan et al., 2009). These finding suggests that DNA methylation mediates the effects of early environment in both rodents and humans and points to the possibility of new therapeutic approaches stemming from translational epigenetic research. Indeed, similar processes at comparable epigenetic labile regions could explain why the adult offspring of high and low licking/grooming mothers exhibit widespread differences in hippocampal gene expression and cognitive function (Weaver, Meaney, & Szyf, 2006).

However, this type of research is limited by the inaccessibility of human brain samples. The translational potential of this finding would be greatly enhanced if the relevant epigenetic modification can be measured in an accessible tissue. Examination of blood samples from adult patients with bipolar disorder, who also retrospectively reported on their experiences of childhood abuse and neglect, found that the degree of DNA methylation of the human glucocorticoid receptor gene promoter was strongly positively related to the reported experience of childhood maltreatment decades earlier. For a relationship between a molecular measure and reported historical exposure, the effects size is extraordinarily large. This opens a range of new possibilities: given the large effect size and consistency of this association, measurement of the GR promoter methylation may effectively become a blood test measuring the physiological traces left on the genome by early experiences. Although this blood test cannot replace current methods of diagnosis, this unique and addition information adds to our knowledge of how disease may arise and be manifested throughout life. Near-future research will examine whether this measure adds value over and above simple reporting of early adversities when it comes to predicting important outcomes, such as response to treatment or suicide.

The old adage you are what you eat might be true on more than just a physical level: The food you choose (and even what your parents and grandparents chose) is reflected in your own personal development and risk for disease in adult life (Wells, 2003). Nutrients can reverse or change DNA methylation and histone modifications, thereby modifying the expression of critical genes associated with physiologic and pathologic processes, including embryonic development, aging, and carcinogenesis. It appears that nutrients can influence the epigenome either by directly inhibiting enzymes that catalyze DNA methylation or histone modifications, or by altering the availability of substrates necessary for those enzymatic reactions. For example, rat mothers fed a diet low in methyl group donors during pregnancy produce offspring with reduced DNMT-1 expression, decreased DNA methylation, and increased histone acetylation at promoter regions of specific genes, including the glucocorticoid receptor, and increased gene expression in the liver of juvenile offspring (Lillycrop, Phillips, Jackson, Hanson, & Burdge, 2005) and adult offspring (Lillycrop et al., 2007). These data suggest that early life nutrition has the potential to influence epigenetic programming in the brain not only during early development but also in adult life, thereby modulating health throughout life. In this regard, nutritional epigenetics has been viewed as an attractive tool to prevent pediatric developmental diseases and cancer, as well as to delay aging-associated processes.

The best evidence relating to the impact of adverse environmental conditions development and health comes from studies of the children of women who were pregnant during two civilian famines of World War II: the Siege of Leningrad (194144) (Bateson, 2001) and the Dutch Hunger Winter (19441945) (Stanner et al., 1997). In the Netherlands famine, women who were previously well nourished were subjected to low caloric intake and associated environmental stressors. Women who endured the famine in the late stages of pregnancy gave birth to smaller babies (Lumey & Stein, 1997) and these children had an increased risk of insulin resistance later in life (Painter, Roseboom, & Bleker, 2005). In addition, offspring who were starved prenatally later experienced impaired glucose tolerance in adulthood, even when food was more abundant (Stanner et al., 1997). Famine exposure at various stages of gestation was associated with a wide range of risks such as increased obesity, higher rates of coronary heart disease, and lower birth weight (Lumey & Stein, 1997). Interestingly, when examined 60 years later, people exposed to famine prenatally showed reduced DNA methylation compared with their unexposed same-sex siblings (Heijmans et al., 2008).

Memories are recollections of actual events stored within our brains. But how is our brain able to form and store these memories? Epigenetic mechanisms inuence genomic activities in the brain to produce long-term changes in synaptic signaling, organization, and morphology, which in turn support learning and memory (Day & Sweatt, 2011).

Neuronal activity in the hippocampus of mice is associated with changes in DNA methylation (Guo et al., 2011), and disruption to genes encoding the DNA methylation machinery cause learning and memory impairments (Feng et al., 2010). DNA methylation has also been implicated in the maintenance of long-term memories, as pharmacological inhibition of DNA methylation and impaired memory (Day & Sweatt, 2011; Miller et al., 2010). These ndings indicate the importance of DNA methylation in mediating synaptic plasticity and cognitive functions, both of which are disturbed in psychological illness.

Changes in histone modications can also inuence long-term memory formation by altering chromatin accessibility and the expression of genes relevant to learning and memory. Memory formation and the associated enhancements in synaptic transmission are accompanied by increases in histone acetylation (Guan et al., 2002) and alterations in histone methylation (Schaefer et al., 2009), which promote gene expression. Conversely, a neuronal increase in histone deacetylase activity, which promotes gene silencing, results in reduced synaptic plasticity and impairs memory (Guan et al., 2009). Pharmacological inhibition of histone deacetylases augments memory formation (Guan et al., 2009; Levenson et al., 2004), further suggesting that histone (de)acetylation regulates this process.

In humans genetic defects in genes encoding the DNA methylation and chromatin machinery exhibit profound effects on cognitive function and mental health (Jiang, Bressler, & Beaudet, 2004). The two best-characterized examples are Rett syndrome (Amir et al., 1999) and Rubinstein-Taybi syndrome (RTS) (Alarcon et al., 2004), which are profound intellectual disability disorders. Both MECP2 and CBP are highly expressed in neurons and are involved in regulating neural gene expression (Chen et al., 2003; Martinowich et al., 2003).

Rett syndrome patients have a mutation in their DNA sequence in a gene called MECP2. MECP2 plays many important roles within the cell: One of these roles is to read the DNA sequence, checking for DNA methylation, and to bind to areas that contain methylation, thereby preventing the wrong proteins from being present. Other roles for MECP2 include promoting the presence of particular, necessary, proteins, ensuring that DNA is packaged properly within the cell and assisting with the production of proteins. MECP2 function also influences gene expression that supports dendritic and synaptic development and hippocampus-dependent memory (Li, Zhong, Chau, Williams, & Chang, 2011; Skene et al., 2010). Mice with altered MECP2 expression exhibit genome-wide increases in histone acetylation, neuron cell death, increased anxiety, cognitive deficits, and social withdrawal (Shahbazian et al., 2002). These findings support a model in which DNA methylation and MECP2 constitute a cell-specific epigenetic mechanism for regulation of histone modification and gene expression, which may be disrupted in Rett syndrome.

RTS patients have a mutation in their DNA sequence in a gene called CBP. One of these roles of CBP is to bind to specific histones and promote histone acetylation, thereby promoting gene expression. Consistent with this function, RTS patients exhibit a genome-wide decrease in histone acetylation and cognitive dysfunction in adulthood (Kalkhoven et al., 2003). The learning and memory deficits are attributed to disrupted neural plasticity (Korzus, Rosenfeld, & Mayford, 2004). Similar to RTS in humans, mice with a mutation of CBP perform poorly in cognitive tasks and show decreased genome-wide histone acetylation (for review, see Josselyn, 2005). In the mouse brain CBP was found to act as an epigenetic switch to promote the birth of new neurons in the brain. Interestingly, this epigenetic mechanism is disrupted in the fetal brains of mice with a mutation of CBP, which, as pups, exhibit early behavioral deficits following removal and separation from their mother (Wang et al., 2010). These findings provide a novel mechanism whereby environmental cues, acting through histone modifying enzymes, can regulate epigenetic status and thereby directly promote neurogenesis, which regulates neurobehavioral development.

Together, these studies demonstrate that misregulation of epigenetic modications and their regulatory enzymes is capable of orchestrating prominent decits in neuronal plasticity and cognitive function. Knowledge from these studies may provide greater insight into other mental disorders such as depression and suicidal behaviors.

Epigenome-wide studies have identied several dozen sites with DNA methylation alterations in genes involved in brain development and neurotransmitter pathways, which had previously been associated with mental illness (Mill et al., 2008). These disorders are complex and typically start at a young age and cause lifelong disability. Often, limited benefits from treatment make these diseases some of the most burdensome disorders for individuals, families, and society. It has become evident that the efforts to identify the primary causes of complex psychiatric disorders may significantly benefit from studies linking environmental effects with changes observed within the individual cells.

Epigenetic events that alter chromatin structure to regulate programs of gene expression have been associated with depression-related behavior and action of antidepressant medications, with increasing evidence for similar mechanisms occurring in post-mortem brains of depressed individuals. In mice, social avoidance resulted in decreased expression of hippocampal genes important in mediating depressive responses (Tsankova et al., 2006). Similarly, chronic social defeat stress was found to decrease expression of genes implicated in normal emotion processing (Lutter et al., 2008). Consistent with these findings, levels of histone markers of increased gene expression were down regulated in human post-mortem brain samples from individuals with a history of clinical depression (Covington et al., 2009).

Administration of antidepressants increased histone markers of increased gene expression and reversed the gene repression induced by defeat stress (Lee, Wynder, Schmidt, McCafferty, & Shiekhattar, 2006; Tsankova et al., 2006; Wilkinson et al., 2009). These results provide support for the use of HDAC inhibitors against depression. Accordingly, several HDAC inhibitors have been found to exert antidepressant effects by each modifying distinct cellular targets (Cassel et al., 2006; Schroeder, Lin, Crusio, & Akbarian, 2007).

There is also increasing evidence that aberrant gene expression resulting from altered epigenetic regulation is associated with the pathophysiology of suicide (McGowan et al., 2008; Poulter et al., 2008). Thus, it is tempting to speculate that there is an epigenetically determined reduced capacity for gene expression, which is required for learning and memory, in the brains of suicide victims.

While the cellular and molecular mechanisms that influence on physical and mental health have long been a central focus of neuroscience, only in recent years has attention turned to the epigenetic mechanisms behind the dynamic changes in gene expression responsible for normal cognitive function and increased risk for mental illness. The links between early environment and epigenetic modifications suggest a mechanism underlying gene-environment interactions. Early environmental adversity alone is not a sufficient cause of mental illness, because many individuals with a history of severe childhood maltreatment or trauma remain healthy. It is increasingly becoming evident that inherited differences in the segments of specific genes may moderate the effects of adversity and determine who is sensitive and who is resilient through a gene-environment interplay. Genes such as the glucocorticoid receptor appear to moderate the effects of childhood adversity on mental illness. Remarkably, epigenetic DNA modifications have been identified that may underlie the long-lasting effects of environment on biological functions. This new epigenetic research is pointing to a new strategy to understanding gene-environment interactions.

The next decade of research will show if this potential can be exploited in the development of new therapeutic options that may alter the traces that early environment leaves on the genome. However, as discussed in this module, the epigenome is not static and can be molded by developmental signals, environmental perturbations, and disease states, which present an experimental challenge in the search for epigenetic risk factors in psychological disorders (Rakyan, Down, Balding, & Beck, 2011). The sample size and epigenomic assay required is dependent on the number of tissues affected, as well as the type and distribution of epigenetic modications. The combination of genetic association maps studies with epigenome-wide developmental studies may help identify novel molecular mechanisms to explain features of inheritance of personality traits and transform our understanding of the biological basis of psychology. Importantly, these epigenetic studies may lead to identification of novel therapeutic targets and enable the development of improved strategies for early diagnosis, prevention, and better treatment of psychological and behavioral disorders.

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Epigenetics in Psychology | Noba

Semin Reprod Med. Author manuscript; available in PMC 2009 Dec 10.

Published in final edited form as:

PMCID: PMC2791696

NIHMSID: NIHMS160913

1 Departments of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan

2 Department of Physiology, School of Medicine, Wayne State University, Detroit, Michigan

1 Departments of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan

3 Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland

1 Departments of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan

2 Department of Physiology, School of Medicine, Wayne State University, Detroit, Michigan

1 Departments of Obstetrics & Gynecology, Wayne State University, Detroit, Michigan

2 Department of Physiology, School of Medicine, Wayne State University, Detroit, Michigan

3 Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland

A vast array of successive epigenetic modifications ensures the creation of a healthy individual. Crucial epigenetic reprogramming events occur during germ cell development and early embryogenesis in mammals. As highlighted by the large offspring syndrome with in vitro conceived ovine and bovine animals, any disturbance during germ cell development or early embryogenesis has the potential to alter epigenetic reprogramming. Therefore the complete array of human assisted reproductive technology (ART), starting from ovarian hormonal stimulation to embryo uterine transfer, could have a profound impact on the epigenetic state of human in vitro produced individuals. Although some investigators have suggested an increased incidence of epigenetic abnormalities in in vitro conceived children, other researchers have refuted these allegations. To date, multiple reasons can be hypothesized why irrefutable epigenetic alterations as a result of ART have not been demonstrated yet.

Keywords: Epigenetics, X-chromosome inactivation, imprinting, transgenerational inheritance

Conrad Waddington introduced the term epigenetics in the early 1940s.1 He defined epigenetics as the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being.2 In the original sense of this definition, epigenetics referred to all molecular pathways modulating the expression of a genotype into a particular phenotype. Over the following years, with the rapid growth of genetics, the meaning of the word has gradually narrowed. Epigenetics has been defined and today is generally accepted as the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence.3 The epigenetic modifications described in current literature generally comprise histone variants, posttranslational modifications of amino acids on the amino-terminal tail of histones, and covalent modifications of DNA bases. The validity of the current definition of epigenetics should be seriously questioned because the previously mentioned epigenetic modifications also have a crucial role in the silencing and expression of noncoding sequences.

In addition to their importance in the commitment of cells to a particular mitotically inheritable form or function, epigenetic marks have a crucial role in guaranteeing genomic stability. Indeed, the silencing of centromeres, telomeres, and transposable elements (TEs) ensures the correct attachment of microtubules to centromeres, reduces excessive recombination between repetitive elements, and prevents transposition of TEs and resulting insertional mutagenesis.46

Although covalent modifications of DNA bases have been described since 1948,7 it was only in 1969 that Griffith and Mahler suggested that these modifications may modulate gene expression.8 The predominant modification in mammalian DNA is methylation of cytosine,7 followed by adenine and guanine methylation.7,9 Although methylation of cytosine bases in mammalian DNA has been primarily described in the context of CpG dinucleotides,10 evidence suggests that cytosines in non-CpG sequences are also frequently methylated.1113 Because the promoter regions of silenced genes possess significantly more methylated cytosines in comparison with actively transcribed genes, this modification has been implicated in transcriptional repression.14,15 Methylation of cytosine in the promoter region may repress gene expression by preventing the binding of specific transcription factors16 or may attract mediators of chromatin remodeling, such as histone-modifying enzymes or other repressors of gene expression.1720 In mammals, the mitotic inheritance of methylated DNA bases is primarily ensured by a maintenance of DNA methyltransferase (DNMT1),2123 whereas DNA methylation enzymes DNMT3A and DNMT3B are mainly responsible for de novo methylation of unmethylated sites.24 Various studies have shown that DNMT3A and DNMT3B target different sites for methylation depending on the cell type and the stage of development.6,25,26 De novo methyltransferases may be directly targeted to specific DNA sequences, may necessitate the interaction with other DNA binding proteins or may be guided by RNA interference (RNAi) in a process called RNA-directed DNA methylation (RdDM).27

Besides covalent modifications of DNA, histones and their posttranslational modifications have also been implicated in the organization of chromatin structure and regulation of gene transcription. Generally, histone classifications comprise the main histones or their variants H1, H2A, H2B, H3, and H4.2831 The fundamental building block of chromatin is the nucleosome and consists of DNA spooled around an octamer of histones. Each octamer contains two units of each principal or variant histone H2A, H2B, H3, and H4.32 Linker DNA connecting nucleosomes associates with the main form or variants of the linker histone H1. A variety of histone-modifying enzymes is responsible for a multiplicity of posttranslational modifications on specific serine, lysine, and arginine residues on the amino-terminal tail of these histones.33,34

The correlation of specific posttranslational modifications on the histones with transcriptional events has resulted in the histone code hypothesis.35 To date, the best characterized modifications are acetylations and methylations of lysine residues on histones H3 and H4. Although all acetylations of lysine residues on H3 and H4 have been associated with transcriptional activation (H3K9, H3K14, H3K18, H3K23, H4K5, H4K8, H4K12, and H4K16),3641 methylation of lysine residues may be either associated with transcriptional repression (H3K9, H3K27, and H4K20) or activation (H3K4, H3K36, and H3K79) depending on which amino acid and to what extent (monomethylation, dimethylation, or trimethylation) the residue is modi-fied.41 Although not as well documented, it has become clear that posttranslational modifications of other histones also have an important role in chromatin structure and gene regulation. Indeed, more recently it has been reported that mutations on specific sites of histones H2A and H2B modify the transcription of various genes.42,43 Similarly, as for DNA methylation enzymes, histone-modifying enzymes may be targeted to specific DNA sequences directly19,20 or may necessitate the interaction of intermediates such as Polycomb and Trithorax group proteins and/or RNAi.4447 In contrast to DNA methylation, it is unclear how and if histone modifications are correctly replicated during mitosis. Although a few investigators have claimed that histone complexes are distributed semiconservatively over the replicated genome,48 most researchers have refuted this manner of histone deposition.49 As a result, it should be questioned whether covalent histone modifications and histone variants are epigenetic marks according to the current definition of epigenetics.

During evolution, an alteration or acquisition of a sex-determining gene on one copy of a pair of chromosomes has resulted in the emergence of sex chromosomes. Consequently, the sexes are generally determined by the presence of a hetero- or homomorphic pair of allosomes. With time, as a result of reduced recombination events between these heteromorphic chromosomes, vastly dissimilar sex chromosomes have arisen. This dissimilarity between the allosomes is at the origin of a gene dosage inequality between the two different sexes.50 To remediate to this imbalance, many species have adopted gene dosage compensation mechanisms. The epigenetic gene dosage compensation mechanisms of genes located on the sex chromosomes vary with species, from simple transcriptional modulation to the entire silencing of one allosome.51 Although it is generally accepted that therian mammals (placentals and marsupials) equalize X-chromosome gene dosage between the sexes by inactivating one X chromosome in females, it has also been suggested that transcription from the active X chromosome is upregulated to maintain balance between autosomal and allosomal gene expression.52 Initially, the observation that female mice heterozygous for X-chromosome-linked coat color genes displayed a mosaic phenotype led to Mary Lyons hypothesis that either the paternally or maternally derived X chromosome could be inactivated in female animals.53 Later investigations revealed that this pattern of X-chromosome inactivation may differ depending on the species and the developmental status of the conceptus. Indeed, female offspring from placentals always possess a mixture of cells with an inactive X chromosome from either maternal or paternal origin, whereas marsupial offspring only present inactive X chromosomes from paternal origin.54,55 In addition, though random X-chromosome inactivation is reported in embryonic lineages from mouse postimplantation embryos, the paternally inherited X-chromosome is always preferentially silenced in preimplantation embryos56 and the resulting extraembryonic lineages.57 This latter form of X-chromosome inactivation is commonly referred to as imprinted X-chromosome inactivation. Although the ultimate outcome of both random and imprinted X-chromosome inactivation is the silencing of one X chromosome, studies suggest that the maintenance of epigenetic marks on the inactive X chromosome is markedly determined by whether the X chromosome underwent random or imprinted inactivation. Indeed, the silencing of imprinted inactive X chromosomes mainly depends on histone modifications applied by Polycomb proteins rather than DNA methylation, whereas DNA methylation is a crucial factor for the maintenance of the inactive state of randomly inactivated X chromosomes.58,59 To date no conclusive evidence exists for imprinted X-chromosome inactivation in human conceptuses.50

To permit random X-chromosome inactivation in the embryonic lineage of mice, a reactivation of the initially silenced X chromosome is necessary. Random X-chromosome inactivation is controlled by a region on the X chromosome called the X inactivation center (XIC). The XIC possesses the genes Xist and Tsix, which contain noncoding RNAs that are crucial for inactivating and maintaining activity of specific X chromosomes. Indeed, transcription of Xist on the inactive X chromosome mediates its silencing, whereas Tsix transcription from the active X chromosome prevents its inactivation.60 Although it remains unknown how X chromosomes are randomly selected for activity or inactivity, three mechanisms have been proposed for the selective silencing of the paternally derived X chromosome during early fetal development. Conceptually, the paternal X chromosome can enter the oocyte in a preinactivated condition or may be selectively silenced after fertilization.51 Meiotic sex chromosome inactivation (MSCI) during spermatogenesis supports the view that the paternal X chromosome can be inherited in an inactive state.61 However, it has also been claimed that MSCI is not crucial for imprinted X-chromosome inactivation because autosomes that do not undergo MSCI, but present Xist transgenes, are also preferentially silenced when paternally inherited.62 In opposition to the inheritance of a preinactivated X chromosome, the differential remodeling of the paternal and maternal chromatin and/or the translation of specific parental imprints on the X chromosomes after fertilization may be at the origin of the initial selective inactivation of the paternal X chromosome in female embryos. Indeed, Xist transcription may be instigated on the paternally derived X-chromosome as a result of the exchange of protamines in the paternal pronucleus with histone variants favoring transcription.63 Alternatively, imprinted X-chromosome inactivation has also been shown to be dependent on various differential epigenetic imprints on Xist and Tsix genes acquired during male and female germ cell development.64,65 In brief, X-chromosome inactivation in mammals has originated to compensate a gene dosage inequality between the two different sexes. Because of its necessity, the establishment and maintenance of X-chromosome inactivation seems to be controlled by a variety of redundant epigenetic marks and mechanisms.

Pronuclear transfer experiments in the early 1980s revealed that mammalian reproduction necessitates the contribution of a paternal and maternal genome to be successful.66,67 The preferential mono-allelic expression of specific genes from either the maternal or paternal allele was believed to be at the origin of this phenomenon. The first imprinted genes in mammals were identified in the early 1990s.6870 Genomic imprinting has been observed in angiosperms and mammals and would have independently evolved in these two taxa as a result of selective pressure on specific genes.71 Although many genes remain imprinted throughout the entire life of an organism, some genes are imprinted in a tissue-specific or temporal manner, similarly to the Xist gene. Imprinted genes are organized in clusters or domains, and their expression is under control of a cis-acting imprinting control element (ICE).72 Similarly to the XIC region on the X chromosome, ICE elements on autosomes acquire differential imprints during germ cell development, depending on their parental origin. Like X-chromosome imprints, autosomal imprints in female mammals are established during folliculogenesis, whereas imprints in males are reset during fetal development.7378 The fact that the imprinted inactivation of the paternal X chromosome and autosomal genes present many molecular similarities has led to the hypothesis that these phenomena have coevolved.79

Although the maintenance, as well as the erasure, of acquired epigenetic marks between generations has both beneficial and deleterious effects, it is unknown to what extent epigenetic marks are maintained or erased between generations in mammals. Because primordial germ cells are set aside during mammalian fetal development and because of epigenetic reprogramming events during germ cell development and early embryogenesis, acquired epigenetic states are believed to be rarely passed on to progeny.80 The erasure of epigenetic marks occurs in female and male mammals during primordial germ cell development and early embryogenesis, whereas the acquisition of epigenetic marks takes place at different times during female and male gametogenesis. Indeed, epigenetic marks in female germ cells are established during folliculogenesis, whereas male germ cells acquire their epigenetic marks during fetal development.7378 The fact that imprints are maintained during early embryogenesis highlights that some sequences may escape reprogramming events. Stella is among a group of proteins that may play an important role in the suppression of epigenetic reprogramming of these specific sequences.81 The failure to erase epigenetic marks during primordial germ cell development or subsequent early embryogenesis is at the origin of transgenerational inheritance of epigenetic traits. A clear example of a gene susceptible to transgenerational inheritance is the Agouti viable yellow (Avy) allele in mice.82 The variable epigenetic status of an intracisternal A particle element (IAP) located upstream from the coding region of Avy in mice is responsible for the variable expression of this allele in adult mice. As a result of incomplete erasure of epigenetic marks on IAPs, this variable expression is often transgenerationally inherited by offspring.82 Evidence suggests that many IAPs fail to undergo epigenetic reprogramming during germ cell development.83 The high incidence of IAPs in mammalian genomes has consequently led to the belief that this type of transgenerational inheritance may be more prevalent than initially conceived.

Given the extent of epigenetic reprogramming that occurs during gametogenesis and embryogenesis and the vulnerability of the process, it is not difficult to understand how alteration in reprogramming could be of clinical relevance. Because epigenetic reprogramming occurs during folliculogenesis and embryogenesis, any disturbance of the normal natural environment during these critical phases could cause epigenetic alterations. Accordingly, researchers have attempted to determine whether children conceived using assistive reproductive technology (ART) carry epigenetic reprogramming defects. A review of an association of ART and epigenetic alterations is covered in detail in articles later in this issue. Importantly, although the whole genome is reprogrammed during germ cell development and embryogenesis, it should be noted that to date only a limited number of loci have been investigated. These loci generally comprise genes in which their epigenetic status significantly affects a perceptible phenotype. Although a specific clinical phenotype has not yet been associated with an epigenetic change, it is it possible that pathology may emerge from a not yet recognized epigenetic alteration.84 An excess of epigenetic alterations could have an immediate impact that precipitates pre- or postnatal death.

At the other extreme, an epigenetic change might result in a perceptible alteration later in life such as cancer, coronary heart disease, stroke, or diabetes. An increased risk of heart disease, stroke, and diabetes is associated with malnutrition in utero and low birth-weight.85 Again, the role of nutrition and diet during pregnancy is covered in detail in ensuing articles in this issue, but it must be considered whether children of ART with a low birthweight could have a predisposition for these chronic phenotypes. Concerns have also been raised about the epigenetic status of tumor suppressors or fertility concerns in individuals exposed to environmental toxins. Subsequent articles address this issue in greater depth as well, but there is sufficient evidence in animals to warrant concern.

In conclusion, there is reason to suspect that early development is vulnerable to unwanted changes in epigenetic inheritance. Animal studies have shown that epigenetic reprogramming is a fragile process that is easily modified,8691 and such data provide compelling biologic plausibility for clinical concern. Although animal models may provide some information, the results may not always be representative of the epigenetic events that occur in humans. Because of the potential for adverse health effects in offspring conceived using ART and in children born from altered nutritional states in pregnancy or exposed to environmental toxins, further research is needed.

This study was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, DHHS (1R03HD046553, 1R21RR021881, and RO1HD045966, and the Reproductive Biology and Medicine Branch, NICHD).

Epigenetics in Reproduction; Guest Editors, James H. Segars, Jr., M.D., and Kjersti M. Aagaard-Tillery, M.D., Ph.D.

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Epigenetics: Definition, Mechanisms and Clinical Perspective